Modified oligonucleotides and methods for their synthesis

ABSTRACT

The present invention is directed to modified oligonucleotides having the following Formula (I)wherein one or more of phosphate groups are substituted at phosphorus, and methods for their synthesis. The modified oligonucleotides are useful for diagnostic applications involving sequence specific recognition of a nucleic acid and for use in a method of medical treatment.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national stage of International Application No.PCT/RU2014/000647, which was filed on Aug. 28, 2014, and which claimspriority to RU 2014134380 which was filed on Aug. 22, 2014, and whichare both herein incorporated by reference.

REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEB

This application includes an electronically submitted sequence listingin .txt format. The .txt file contains a sequence listing entitled“6611_0005PUS1_ST25.txt” created on Nov. 12, 2019 and is 17,281 bytes insize. The sequence listing contained in this .txt file is part of thespecification and is hereby incorporated by reference herein in itsentirety.

TECHNICAL FIELD

The present invention relates to nucleotides and oligonucleotides havinga modified phosphate group, and methods for their synthesis.

BACKGROUND

Nucleic acid derivatives such as oligonucleotides appended with variousadditional functionalities are used widely as research tools in lifesciences, in particular, they are regarded as promising therapeutics [1]and sensitive probes for molecular diagnostics [2]. Severaloligonucleotide therapeutics have received FDA approval to go intoclinic. Examples include an antiviral agent Vitravene (Fomivirsen, ISIS2922) [3], an anti-angiogenic aptamer Macugen (Pegaptanib sodium) [4]and an anti-cholesterol gapmer Mipomersen (Kynamro, ISIS 301012) [5]. Anumber of other oligonucleotide candidates such as siRNA, DNAzymes andantisense morpholino analogues (PMO) are currently undergoing variousphases of clinical trials.

To be regarded as potential therapeutic candidates, oligonucleotidesshould correspond to the following requirements.

-   1. Sufficient stability and sequence-specificity of a complementary    complex with their biological target (most often it is a cellular    RNA).-   2. Increased resistance in biological media such as serum.-   3. Beneficial physico-chemical properties such as aqueous solubility    and chemical stability.-   4. Cost-effective synthesis and affordable price.-   5. Efficient cell uptake and in vivo delivery, preferably in the    absence of external transfection agents.

According to the mechanism of action, oligonucleotide analogues caninterfere with practically any stage of genetic information transfer:either from DNA to RNA (transcription) or from RNA to protein(translation). Inhibition of transcription is performed by bindinggenomic DNA by triplex-forming oligonucleotides (TFOs) [6], inparticular, peptide nucleic acids (PNAs) [7]. Inhibition of translation(antisense mechanism) is realised through mRNA blocking [8]. Most ofknown to-date oligonucleotide analogues act by antisense mechanism.Those are small interfering RNAs (siRNAs) [9], nucleic acid enzymes(ribozymes or DNAzymes) [10], and a majority of chemically modifiedoligonucleotide analogues [11]. Specific oligonucleotide derivativessuch as aptamers can also block protein function by direct binding toproteins or small molecule co-factors [12].

Most of antisense oligonucleotide analogues bind mRNA and inhibittranslation via steric block [13]. Those include a majority of analogueswith modifications in the sugar such as 2′-fluoro[14], 2′-O-methyl [15],2′-O-β-methoxyethyl (2′-MOE) [16] or locked nucleic acid (LNA) [17]derivatives. Oligonucleotide analogues, which substitute an unchargedgroup for anionic internucleoside phosphate group such asmethylphosphonates [18], phosphotriesters [19] or phosphoramidates [20]also act by steric block. The same antisense mechanism is involved inaction of distant nucleic acid mimics such as PNAs [21] orphosphorodiamidate morpholino oligonucleotides (PMO) [22].

Additional interest attract those analogues, which are able toirreversibly inactivate RNA by catalysing its hydrolytic cleavage, forexample, via recruiting cellular enzyme RNase H by 2′-deoxyphosphorothioates [23], ara-2′-fluoro derivatives (2′-FANA) [24] orgapmers [24]. SiRNAs induce catalytic cleavage of mRNA by activatingRISC complex with ribonuclease activity [25] whereas nucleic acidenzymes (ribozymes or DNAzymes) do not require proteins for theircatalytic RNA-cleaving action [27].

Many oligonucleotide analogues have modified internucleoside phosphategroups. Among them phosphorothioates [28], phosphorodithioates [29] andboranophosphates [30]. A positive feature of those derivatives is theirrelatively low cost due to the use of natural 2′-deoxyribonucleotidesand highly effective solid-phase DNA phosphoramidite chemistry [31].Phosphate-modified analogues contain asymmetric phosphorus atom(s) andare usually obtained as a mixture of 2^(n-1) diastereomers for n-meroligonucleotide. Different diastereomers often have different affinityto RNA and enzyme resistance, which are crucial for potential antisenseaction.

Currently, a task of especial priority is the development ofoligonucleotide analogues with efficient cell uptake and in vivodelivery, preferably in the absence of external delivery agents such ascationic lipids, polymers or nanoparticles. Here, oligonucleotideanalogues with reduced or completely eliminated negative charge may beparticularly interesting [32]. Among them are oligonucleosidephosphoramidates that substitute charge neutral phosphoramidate groupfor anionic phosphate. Chemical synthesis of phosphoramidates isrelatively straightforward. However, those analogues exhibit reducedaffinity to RNA [33] and are sensitive to acidic hydrolysis [34].N3′→P5′ phosphoramidates have improved RNA binding but are difficult tosynthesise [35]. Those representatives that have positively chargedgroups in the side chains are more accessible and have excellentenzymatic stability but their affinity to RNA is lower [36]. At the sametime a majority of known phosphoramidate derivatives including suchuseful antisense agents as morpholinos (PMOs) [37] show some lability atacidic pH. Improved acid stability would be required to preventdegradation of oligonucleotide analogues inside endosomes.

Over the last decade new phosphonate oligonucleotide analogues haveemerged, which substitute an ionisable phosphonate group for naturalphosphate. Among them are phosphonoacetates and thiophosphonoacetates[38], phosphonoformates [39] and 1,2,3-triazol-4-ylphosphonates [40].Those compounds exhibit increased biological resistance and adequate RNAbinding, and, additionally, they support RNase H cleavage and haveimproved cell uptake even in the absence of transfection agents.However, their chemical synthesis is difficult and costly.

Modified nucleotides and oligonuclides containing at least once thestructure P=N-Acc, wherein Acc is an electronic acceptor have also beendescribed [41]. Suitable identities for Acc are —CN, —SO₂R andelectron-deficient, six membered N⁺ heterocycles in which at least onenitrogen is alkylated and in an ortho or para position.

At the moment, considerable attention is drawn to phosphorodiamidatemorpholino oligonucleotides (PMOs), which are known antisense agents[42]. They are commercially available from GeneTools LLC. PMOs areactively explored as potential therapeutics by Sarepta Therapeutics,(until 2012 AVI Biopharma). In 2013 the company announced successfulcompletion of Phase III clinical trials aganst Duchenne musculardystrophy (DMD) by a PMO drug Eteplirsen (AVI-4658), which correctsaberrant splicing of dystrophin pre-mRNA [43]. At the beginning of 2014Sarepta Therapeutics has said that their morpholino drug candidateAVI-7288 has successfully passed Phase I clinical trials against deadlyMarburg hemorrhagic fever caused by an RNA-containing virus [44].

However, morpholinos are acid-sensitive just as other phosphoramidates[45]. Moreover, their synthesis is based on P(V) chemistry [46]. Thechemistry may lead to side-reactions such as the modification of the O⁶in guanine [47]. It can be prevented by a protecting group at the O⁶position [48] but that requires a special G monomer, which adds up tothe costs of PMO synthesis. Another drawback of the chemistry is that itis incompatible with common phosphoramidite method and cannot use themodifying and labeling reagents available from usual suppliers such asGlen Research, Inc.

Another serious handicap of PMOs is the difficulty of their chemicalmodification to obtain various derivatives for structure-activitystudies. Only a few side-chain modifications to PMO were proposed thatare claimed to enhance their cell uptake and therapeutic efficacy [49].

Morpholinos often show relatively poor efficiencies of cell uptake andhence high repeated doses are required for good therapeutic effect to beseen. Cell uptake of PMO-peptide conjugates is much higher than fornaked PMO and thus much lower doses are needed in vivo [50]. There is aneed to obtain better oligonucleotide analogues that will show greaterlevels of cell and tissue delivery and improved therapeutic efficacy inthe absence of delivery aids.

Thus, the development of new oligonucleotide analogues remains animportant task.

SUMMARY OF THE INVENTION

This present invention is based on the inventors' insight intoprospective candidates for oligonucleotide therapeutics with improvedcell uptake. Specifically, the present invention is based on thefollowing ideas for suitable approaches to/characteristics for new sucholigonucleotide therapeutics:

-   1. Substitute charge neutral or positively charged groups for    natural anionic phosphate.-   2. Comply with the requirements for potential oligonucleotide    therapeutics outlined above.-   3. Preferably retain a conventional nucleotide backbone.-   4. Display sufficient chemical stability.-   5. Possess structural flexibility that allows for incorporation of    diverse side-chain groups.-   6. Have low toxicity.

New oligonucleotide analogues that correspond to the above requirementsand may potentially exhibit improved cell uptake are disclosed herein.Broadly speaking, these oligonucleotide analogues are in the class ofphosphoryl imines and analogues thereof, for example, phosphorylguanidines, phosphoryl amidines, phosphoryl isoureas, phosphorylisothioureas, phosphoryl imidates, phosphoryl imidothioates andanalogues thereof.

Accordingly, in one aspect the present invention relates to a nucleotideor oligonucleotide comprising a phosphoryl guanidine (FI), phosphorylamidine (FII), phosphoryl isourea (FIII), phosphoryl isothiourea (FIV),phosphoryl imidate (FV) or phosphoryl imidothioate (FVI), for example, aphosphoryl guanidine (FI), phosphoryl amidine (FII), phosphoryl isourea(FIII), phosphoryl isothiourea (FIV). It will be appreciated that theinvention also relates to modified phosphate versions of these moieties;that is, phosphates in which one or more of the oxygen atoms surroundingthe phosphorus atoms has been replaced, for example, by a sulfur atom,selenium atom, imino group or a borane.

For example, the present invention relates to, inter alia, nucleotidesor oligonucleotides, and analogues thereof, comprising one or more ofthe following motifs:

wherein ---- indicates a point of attachment to a substituent.

These modified phosphate groups are of considerable interest because oftheir physico-chemical properties. Without wishing to be bound to anyparticular theory, the present inventors believe that these moieties canbe charge neutral, charge negative or charge positive at physiologicalpH, making them of considerable interest for the synthesis ofnucleotides and oligonucleotides useful in therapeutic, diagnostic andresearch applications.

Furthermore, the present inventors have found that these modifiedphosphate groups can be incorporated into oligonucleotide structuresconveniently using methods as described herein, and are compatible withmany known and biologically interesting modified and/or labelledoligonucleotide motifs and sequences.

For example, the motif may be:

wherein:R¹ is selectedfrom —NR^(1A)R^(1B), —OR³, —SR³, —H, —S(O)H, —S(O)R³, —S(O)₂H, —S(O)₂R³,—S(O)₂NH₂, —S(O)₂N HR³, —S(O)₂NR³ ₂, —C₂₋₁₀alkenyl, —C₂₋₁₀alkynyl,—C₆₋₁₀aryl, or —O₅₋₁₀heteroaryl;R² is selected from —H, —NR^(2A)R^(2B), —OR³, —SR³,halogen, —CN, —S(O)H, —S(O)R³, —S(O)₂H, —S(O)₂R³, —S(O)₂NH₂, —S(O)₂NHR³,—S(O)₂NR³ ₂, C₂₋₁₀alkenyl, C₂₋₁₀alkynyl, —C₆₋₁₀aryl, or—C₅₋₁₀heteroaryl;

-   -   wherein each R^(1A), and R^(2B) is independently selected from        —H, —C₂₋₁₀alkenyl, —C₂₋₁₀alkynyl, —C₆₋₁₀aryl, or        —O₅₋₁₀heteroaryl;    -   optionally wherein R^(1A) and R^(2A) together form an alkylene        or heteroalkylene chain of 2-4 atoms in length;    -   optionally wherein R^(1A) and R^(1B), together with the atom to        which they are bound, form a 5-8 membered heterocycle;    -   optionally wherein R^(2A) and R^(2B), together with the atom to        which they are bound, form a 5-8 membered heterocycle;        R³ is selected from —C₁₋₁₀alkyl, —C₂₋₁₀alkenyl, —C₂₋₁₀alkynyl,        —C₆₋₁₀aryl, or —O₅₋₁₀heteroaryl;    -   wherein each alkyl, alkenyl, alkynyl, aryl, heteroaryl, alkylene        or heteroalkylene is optionally substituted.

Accordingly, in a first aspect, the present invention may provide acompound of Formula (I):

wherein Z is selected from —O⁻, —S⁻, —Se⁻, —N⁻R^(N), or —BH₃ ⁻, whereinR^(N) is H, C₁₋₄alkyl, or a protecting group;X is selected from the 5′ end of a nucleoside, nucleoside analogue,oligonucleotide, or oligonucleotide analogue and Y is selected from the3′ end of a nucleoside, nucleoside analogue, oligonucleotide, oroligonucleotide analogue; —H, —OH, —SH, NHR^(N), —O-PG, or S-PG, whereinPG is a protecting group; a linker, a monophosphate or diphosphate, or alabel or quencher;orY is selected from the 3′ end of a nucleoside, nucleoside analogue,oligonucleotide, or oligonucleotide analogue and X is selected from the5′ end of a nucleoside, nucleoside analogue, oligonucleotide, oroligonucleotide analogue, —H, —OH, —SH, NHR^(N), —O-PG, or S-PG, whereinPG is a protecting group; a linker, a monophosphate or diphosphate, or alabel or quencher;R¹ is selected from —NR^(1A)R^(1B), —OR³, —SR³, —H, —S(O)H, —S(O)R³,—S(O)₂H, —S(O)₂R³, —S(O)₂NH₂, —S(O)₂N HR³, —S(O)₂NR³ ₂, —C₂₋₁₀alkenyl,—C₂₋₁₀alkynyl, —C₆₋₁₀aryl, or —O₅₋₁₀heteroaryl;R² is selected from —H, —NR^(2A)R^(2B), —OR³, —SR³,halogen, —CN, —S(O)H, —S(O)R³, —S(O)₂H, —S(O)₂R³, —S(O)₂NH₂, —S(O)₂NHR³,—S(O)₂NR³ ₂, C₂₋₁₀alkenyl, C₂₋₁₀alkynyl, —C₆₋₁₀aryl, or—C₅₋₁₀heteroaryl;wherein each R^(1A), R^(2A), and R^(2B) is independently selected from—H, —C₁₋₁₀alkyl, —C₂₋₁₀alkenyl, —C₂₋₁₀alkynyl, —C₆₋₁₀aryl, or—C₅₋₁₀heteroaryl;

-   -   optionally wherein R^(1A) and R^(2A) together form an alkylene        or heteroalkylene chain of 2-4 atoms in length;    -   optionally wherein R^(1A) and R^(1B), together with the atom to        which they are bound, form a 5-8 membered heterocycle;    -   optionally wherein R^(2A) and R^(2B), together with the atom to        which they are bound, form a 5-8 membered heterocycle;        R³ is selected from —C₁₋₁₀alkyl, —C₂₋₁₀alkenyl, —C₂₋₁₀alkynyl,        —C₆₋₁₀aryl, or —C₅₋₁₀heteroaryl;        wherein each alkyl, aryl, heteroaryl, alkylene or heteroalkylene        is optionally substituted.

In some embodiments, R¹ is selected from —NR^(1A)R^(1B), —OR³, and —SR³.In some embodiments, R¹ is —NR^(1A)R^(1B).

Accordingly, the compound may be a compound of formula (Ia), wherein X,Y, Z, R^(1A), R^(1B) and R² are as defined herein.

The compound maybe a “modified” nucleotide, “modified” oligonucleotide,or “modified” nucleoside triphosphate. It will be understood that theterm “modified” in this context refers to the incorporation of themodified phosphate moiety depicted in Formula (I), that is, a phosphatecomprising the motif depicted in Formula (FVII).

Nucleoside triphosphates are molecules containing a nucleoside bound tothree phosphate groups. It will be understood that in this context, theterm phosphate includes modified phosphates, as defined herein.Suitably, the nucleoside triphosphate has a triphosphate group that isthree phosphates linked together in a row; that is, the molecule may bea dNTP or similar. In other embodiments, nucleosides may have phosphatesat both the 5′ and 3′ positions, for example, a single phosphate groupat the 5′ position and diphosphate group (two phosphates linkedtogether) at the 3′ position. It will be appreciated that any and all ofthese phosphate groups may be modified as described herein.

When the compound is an oligonucleotide, it will be understood that eachfurther nucleoside may itself independently be a nucleoside analogueand, additionally or alternatively, each further phosphate group (ifpresent) may in itself be modified.

In some embodiments, R² is selected from —H, —NR^(2A)R^(2B), —OR³, —SR³,—S(O)H, —S(O)R³, —S(O)₂H, —S(O)₂R³, —S(O)₂NH₂, —S(O)₂N HR³, —S(O)₂NR³ ₂,—C₂₋₁₀alkenyl, —C₂₋₁₀alkynyl, —C₆₋₁₀aryl, or —O₅₋₁₀heteroaryl, whereineach alkyl, aryl, heteroaryl, alkylene or heteroalkylene is optionallysubstituted.

In some embodiments, R² is —H, —NR^(2A)R^(2B), —OR³, —SR³, C₁₋₁₀alkyl,C₂₋₁₀alkenyl, C₂₋₁₀alkynyl, —C₆₋₁₀aryl, or —C₅₋₁₀heteroaryl, whereineach alkyl, aryl, heteroaryl, alkylene or heteroalkylene is optionallysubstituted.

In some embodiments, R² is —H, —NR^(2A)R^(2B) or —OR³. For example, thecompound may contain a phosphoryl formamidine (P—N═CHNR^(1A)R^(1B))group, a phosphoryl guanidine (P—N═C(NR^(1A)R^(1B)) (NR^(2A)R^(2B))), ora phosphoryl isourea (P—N═C(NR^(1A)R^(1B))OR³) group.

In some embodiments, R³ is C₁₋₄alkyl, preferably methyl.

In some embodiments, R^(1A) is independently selected from —H and—C₁₋₄alkyl optionally substituted with one or more substituents selectedfrom —F, —Cl, —Br, —I, —OH, —C₁₋₄alkoxy, —NH₂, —NH(C₁₋₄alkyl), and—N(C₁₋₄alkyl)₂; preferably from —H and —C₁₋₄alkyl, more preferably from—H and methyl.

In some embodiments, R^(1B) is independently selected from —H and—C₁₋₄alkyl optionally substituted with one or more substituents selectedfrom —F, —Cl, —Br, —I, —OH, —C₁₋₄alkoxy, —NH₂, —NH(C₁₋₄alkyl), and—N(C₁₋₄alkyl)₂; preferably from —H and —C₁₋₄alkyl, more preferably from—H and methyl.

In some embodiments, R^(2A) is independently selected from —H and—C₁₋₄alkyl optionally substituted with one or more substituents selectedfrom —F, —Cl, —Br, —I, —OH, —C₁₋₄alkoxy, —NH₂, —NH(C₁₋₄alkyl), and—N(C₁₋₄alkyl)₂; preferably from —H and —C₁₋₄alkyl, more preferably from—H and methyl.

In some embodiments, R^(2B) is independently selected from —H and—C₁₋₄alkyl optionally substituted with one or more substituents selectedfrom —F, —Cl, —OH, —C₁₋₄alkoxy, —NH(C₁₋₄alkyl), and —N(C₁₋₄alkyl)₂;preferably from —H and —C₁₋₄alkyl, more preferably from —H and methyl.

In some embodiments, each R^(1A), R^(1B), R^(2A), and R^(2B) isindependently selected from —H and —C₁₋₄alkyl optionally substitutedwith one or more substituents selected from —F, —Cl, —Br, —I, —OH,—C₁₋₄alkoxy, —NH₂, —NH(C₁₋₄alkyl), and —N(C₁₋₄alkyl)₂; preferably from—H and —C₁₋₄alkyl optionally substituted with one to three substituentsselected from —F, —Cl, —OH, and —NH₂, more preferably from —H and—C₁₋₄alkyl, more preferably from —H and methyl.

In some embodiments, R² is —NR^(2A)R^(2B), preferably —NMe₂.

In some embodiments, R¹ is —NH₂ or —NMe₂.

In some embodiments, R^(1A) and R^(2A) together form an alkylene orheteroalkylene chain of 2-4 atoms in length and R^(1B) and R^(2B) areeach independently selected from —H and —C₁₋₄alkyl. In some embodiments,R^(1A) and R^(2A) together form —CH₂—CH₂—. In some embodiments, R^(1A)and R^(2A) together form —CH₂—CH₂— and R^(1B) and R^(2B) are —H ormethyl.

In some embodiments, R^(1A) and R^(1B), together with the atom to whichthey are bound, form a 5-8 membered heterocycle, preferably apyrrolidine, piperidine, piperazine or morpholine. Suitably they,together with the atom to which they are bound, form a 5-memberedheterocycle, preferably a pyrrolidine.

In some embodiments, R^(2A) and R^(2B), together with the atom to whichthey are bound, form a 5-8 membered heterocycle, preferably pyrrolidine,piperidine, piperazine or morpholine. Suitably they, together with theatom to which they are bound, form a 5-membered heterocycle, preferablya pyrrolidine.

As described herein, Z may be selected from —O⁻, —S⁻, —Se⁻, —N⁻R^(N), or—BH₃, wherein R^(N) is —H, —C₁₋₄alkyl, or a protecting group.Preferably, Z is from —O⁻ or —S⁻, most preferably —O⁻. It will beappreciated that —P⁺—O⁻ is a resonance structure of —P═O.

Certain preferred compounds are compounds of the following formulae:

In a further aspect, the present invention provides an oligonucleotidehaving at least one modified phosphate moiety of formula FVII:

wherein R¹ and R² are as defined herein.

In a further aspect, the present invention provides an oligonucleotidewherein a phosphate linking adjacent nucleosides/nucleoside analoguescomprises a phosphoryl guanidine, phosphoryl amidine, phosphorylisourea, phosphoryl isothiourea, phosphoryl imidate or phosphorylimidothioate.

In a further aspect, the present invention provides a method ofsynthesizing a compound comprising a motif of Formula FVII:

In one aspect, the method comprises reaction of a phosphorous acidderivative with an imino derivative HN═CR¹R² or an N-silylated iminoderivative R^(Si) ₃SiN═CR¹R² in the presence of an oxidant, andoptionally a silylating agent and/or a base (Procedure A), wherein eachR^(Si) is an alkyl or aryl group. For example, the phosphorous acidderivative may be a phosphite or H-phosphonate.

Examples of imino derivatives for Procedure A include, withoutlimitation, 1,1,3,3-tetramethylguanidine (TMG), guanidine hydrochloride,1,1-dimethylguanidine sulphate, 1,3-diphenylguanidine, formamidinehydrochloride, acetamidine hydrochloride, 1H-pyrazole-1-carboxamidinehydrochloride, N-Boc-1H-pyrazole-1-carboxamidine, ethyl formimidatehydrochloride, ethyl acetimidate hydrochloride, O-methylisourea hydrogensulfate, S-methylisothiourea hydrogen sulphate, S-benzylisothioureahydrochloride and the like.

It will be appreciated that an imino derivative may be in the form of afree base or a salt as described herein. If the imino derivative is asalt, a base may be added to liberate a free base of the iminoderivative and/or a silylating agent may be employed to produce anN-silylated derivative of an imino compound.

The oxidant may be iodine I₂, bromine Br₂, chlorine Cl₂, iodine chlorideICI, N-bromosuccinimide, N-chlorosuccinimide, N-iodosuccinimide, carbontetrachloride CCl₄, bromotrichloromethane CCl₃Br, tetrabromomethaneCBr₄, tetraiodomethane Cl₄, iodoform CHI₃, hexachloroethane C₂Cl₆,hexachloroacetone (CCl₃)₂CO or the like. A preferred oxidant is iodineI₂.

In a further aspect, the method comprises reaction of a phosphorous acidderivative with an organic azide, optionally in the presence of asilylating agent and/or a base (Procedure B).

For example, the organic azide may be selected from a bis(disubstitutedamino)-1-azidocarbenium salt, a 1-(disubstitutedamino)-1-azidocarbamidinium salt, a 1-(disubstitutedamino)-1-azido-ethene, an N-substituted-1-azidocarbamidine and the like.

In the reactions of Procedure A and Procedure B, the reaction may becarried out in the presence of a silylating agent, for example,N,O-bis(trimethylsilyl)acetamide (BSA),N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA),chlorotrimethylsilane, bromotrimethylsilane, iodotrimethylsilane,triethylsilyl chloride, triphenylsilyl chloride, hexamethyldisilazane,trimethylsilyl trifluoromethanesulfonate (TMSOTf),dimethylisopropylsilyl chloride, diethylisopropylsilyl chloride,tert-butyldimethylsilyl chloride, tert-butyldiphenylsilyl chloride,triisopropylsilyl chloride, dimethyldichlorosilane,diphenyldichlorosilane and the like. For example, the reaction may becarried out in the presence of a silylating agent selected fromN,O-bis(trimethylsilyl)acetamide (BSA),N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA), andchlorotrimethylsilane.

In the reactions of Procedure A and Procedure B, the reaction may becarried out in the presence of a base, for example, triethylamine,N,N-diisopropylethylamine (DIEA), N-methylmorpholine, N-ethylmorpholine,tributylamine, 1,4-diazabicyclo[2.2.2]octane (DABCO), N-methylimidazole(NMI), pyridine, 2,6-lutidine, 2,4,6-collidine, 4-dimethylaminopyridine(DMAP), 1,8-bis(dimethylamino)naphthalene (“proton sponge”),1,8-diazabicyclo[5.4.0]undec-7-ene (DBU),1,5-diazabicyclo[4.3.0]non-5-ene (DBN),1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD),7-methyl-1,5,7-triazabicyclo[4.4.0]-dec-5-ene (MTBD),1,1,3,3-tetramethylguanidine (TMG),2-tert-butyl-1,1,3,3-tetramethylguanidine,2,8,9-trimethyl-2,5,8,9-tetraaza-1-phosphabicyclo[3.3.3]-undecane,phosphazene base or the like.

Examples of solvents for Procedure A include, without limitation,pyridine, 2-picoline, 3-picoline, 4-picoline, quinoline, tetrahydrofuran(THF), 1,4-dioxane, 1,2-dimethoxyethane (DME), diethylene glycoldimethyl ether (diglym), diethyl ether, acetonitrile and the like.

Examples of solvents for Procedure B include, without limitation,acetonitrile, N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA),N-methylpyrrolidone (NMP), tetramethyl urea,1,3-dimethylimidazolidin-2-one, sulfolane, hexamethyl phosphortriamide(HMPT), 1,4-dioxane, tetrahydrofuran (THF), acetone, ethyl acetate andthe like.

Further details of these Procedures are provided below.

As described herein, a particular advantage of the modified phosphatemoieties of the present invention is the convenient incorporation ofthese moieties as demonstrated by the present inventors. For example, amodified phosphate moiety of Formula VI can be readily incorporated intoan oligonucleotide during sequential oligonucleotide synthesis based onH-phosphonate or phosphoramidite chemistry. Further details are providedbelow.

Any one or more of the aspects of the present invention may be combinedwith any one or more of the other aspects of the present invention.Similarly, any one or more of the features and optional features of anyof the aspects may be applied to any one of the other aspects. Thus, thediscussion herein of optional and preferred features may apply to someor all of the aspects. In particular, optional and preferred featuresrelating to the compounds, motifs, and intermediates, methods of makingthe compounds and methods of using the compounds, etc apply to all ofthe other aspects. For example, substituents preferences for thecompounds also apply to the azides/imino derivatives and visa versa.Furthermore, optional and preferred features associated with a method oruse may also apply to a product and vice versa.

FIGURES

The invention is further described, without limitation, with referenceto the following:

FIG. 1. RP-HPLC of oligonucleotides 5′-d(GCGCCAAACpA)(SEQ ID NO: 11)(thin black line), 5′-d(GCGCCAAApCpA) (SEQ ID NO: 7) (grey line) and5′-d(GCGCCAApApCpA) (SEQ ID NO: 12) (bold black line) modified withN,N,N′,N′-tetramethyl-N″-phosphorylguanidine groups; p here and belowmarks position of a modifying group.

FIG. 2. RP-HPLC of oligonucleotide 5′-d(TTTTTTTTTTTTTTTTTTTpT) (SEQ IDNO: 13) modified with N-phosphorylguanidine group.

FIG. 3. RP-HPLC of oligodeoxyribonucleotides with an LNA nucleotide5′-d(TTTT)tdT (thin black line), 5′-d(TTTT)tpdT (grey line) and5′-tpd(TTTTT) (dashed line) modified withN,N,N′,N′-tetramethyl-N″-phosphorylguanidine groups; t marks position ofan LNA nucleotide.

FIG. 4. RP-HPLC of oligo-2′-O-methylribonucleotides 5′-UUUUUp*U modifiedwith a N,N′-bis(tetramethylene)-N″-phosphorylguanidine group (bold blackline), 5′-UUUUUpU modified with aN,N′-dimethyl-N″-phosphorylimino-2-imidazolidine group (grey line), andan unmodified oligo-2′-O-methylribonucleotide 5′-UUUUUU (thin blackline).

FIG. 5. RP-UPLC of an oligonucleotide 5′-d(TpTTTTT) modified with aN-cyanoiminophosphate group.

FIG. 6. RP-HPLC of oligonucleotides 5′-d(TTTTTT)p (bold black line),5′-DMTr-Flu pd(TTTTTT) (thin black line) and a nucleotide 5′-DMTr-FlupdT (grey line) modified with aN,N′-bis(tetramethylene)-N″-guanidinophosphate group.

FIG. 7. RP-HPLC of oligonucleotides 5′-d(TTTTTpT), 5′-d(TTTTpTpT),5′-d(TTTpTpTpT), 5′-d(TTpTpTpTpT) and 5′-d(TpTpTpTpTpT) modified withN,N′-dimethyl-N″-phosphorylimino-2-imidazolidine groups; p marksposition of a modifying group.

FIG. 8. RP-HPLC of an oligonucleotide 5′-d(GpCpGpCpCpApApApCpA)(SEQ IDNO: 1) fully modified withN,N′-dimethyl-N″-phosphorylimino-2-imidazolidine groups at allinternucleoside positions.

ABBREVIATIONS AND NOTATION

-   p—indicates the position of a modified phosphate group as described-   t—indicates the position of an LNA-T nucleotide-   a—indicates the position of an LNA-A nucleotide-   c—indicates the position of an LNA-5-Me-C nucleotide-   g—indicates the position of an LNA-G nucleotide-   F—2-hydroxymethyl-3-hydroxytetrahydrofurane (apurinic/apyrimidinic    site) phosphate-   BHQ—BlackHole Quencher™-   DD—1,12-dodecanediol phosphate-   Flu—5(6)-carboxyfluorescein label-   N_(s)—indicates the phosphorothioate residue of nucleotide “N”-   BSA—N,O-bis(trimethylsilyl)acetamide-   BSTFA—N,O-bis(trimethylsilyl)trifluoroacetamide-   DIEA—N,N-diisopropylethylamine-   NMI—N-methylimidazole-   DMAP—4-dimethylaminopyridine-   DBU—1,8-diazabicyclo[5.4.0]undec-7-ene-   TMG—1,1,3,3-tetramethylguanidine

DETAILED DESCRIPTION

Phosphoryl guanidines are represented in Nature by such compounds ascreatine phosphate and phosphoarginine. Synthetic low molecular weightphosphoryl guanidines have been known since at least 1960s [52]. Smallmolecule phosphoryl guanidines have found limited use as pesticides[53], flame retardants [54] and therapeutics [55]. Phosphoryl guanidinediesters form complexes with metal ions through guanidine nitrogen [56].X-ray analysis of O,O′-diisopropyl phosphoryl guanidine has confirmedthe presence of only the tautomer with the phosphoryliminogroup >P(═O)—N═C< in the crystal structure.

However, no nucleoside or oligonucleotide derivatives have beendescribed until now, and their properties remained unknown. The presentinventors have prepared first oligonucleoside phosphoryl guanidines byiodine oxidation of dithymidine β-cyanoethyl phosphite in the presenceof N,N,N′,N′-tetramethylguanidine (TMG) in pyridine using methodologysimilar to that previously described in relation to primary amines [57].

The present inventors have studied oxidation of CPG-bound3′,5′-dithymidine β-cyanoethyl phosphite, which is a common intermediatein solid-phase DNA synthesis according to the β-cyanoethylphosphoramidite method [58]. As described herein, oxidation of the abovephosphite by 0.1 M solution of iodine in dry pyridine in the presence of1 M TMG and 20% N,O-bis(trimethylsilyl)acetamide produced3′,5′-dithymidine-N,N,N′,N′-tetramethyl phosphoryl guanidine as a majorproduct. The oligonucleotide 5′-d(TTTTTpT), where p indicates positionof the modified phosphate group, was isolated after concentrated aqueousammonia treatment for 1 h at ambient temperature as a mixture of twodiastereomers (Example 1.1). The only byproduct that was isolated wasdT₆, which is the likely result of concurrent hydrolysis of a reactiveiodophosphonium intermediate by traces of moisture. The inventors haveconcluded that the phosphoryl guanidine group is stable duringsolid-phase oligonucleotide synthesis and ammonia deprotection at pH 11.

The integrity of the oligothymidine monophosphoryl guanidine wasconfirmed by MALDI-TOF MS. Its mobility during gel electrophoresis in20% polyacrylamide gel was lower than for dT6, suggesting charge neutralcharacter of the tetramethyl phosphoryl guanidine group at pH 7.4, whichcorresponds to the literature data for low molecular weight phosphorylguanidines [59].

Through synthesis of 20-mer oligothymidylates with one tetramethylphosphoryl guanidine group, the inventors have noted that the yieldsprogressively worsen with increased from the support in a row5′-d(T₁₈TpT)>5′-d(T₁₀TpT₉)>5′-d(TTpT₁₈). The oligothymidylates obtainedwere used to ascertain the influence of the single tetramethylphosphoryl guanidine group on thermal stability of the complementaryduplex with oligonucleotide 5′-d(C₂A₂₀C₂) as compared to unmodifieddT₂₀.

The inventors have also successfully obtained oligonucleotides withunsubstituted phosphoryl guanidine group. Oxidation of CPG-bound3′,5′-dithymidine β-cyanoethyl phosphite by 0.1 M iodine solution inpyridine in the presence of 0.5 M guanidine hydrochloride, 0.5 M1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and 20% BSA followed byoligonucleotide synthesis produced a hexathymidylate with phosphorylguanidine group as a major product together with the dT₆ hydrolysisproduct (Example 5).

Analogously, iodine oxidation of the dithymidine phosphite in thepresence of formamidine hydrochloride, DBU and BSA in pyridine followedby oligonucleotide synthesis resulted in the formation of hexathymidinephosphoryl formamidine, which is a representative of another class ofphosphorylimino compounds: phosphoryl amidines (Example 39).

Oxidation of CPG-bound 3′,5′-dithymidine β-cyanoethyl phosphite by 0.1 Miodine solution in pyridine in the presence of 0.5 M O-methylisoureahydrogen sulfate, 1 M DBU and 20% BSA followed by oligonucleotidesynthesis and deprotection by concentrated aqueous ammonia for 1 h atambient temperature has resulted in a mixture of products containingdT₆, oligonucleoside O-methyl phosphoryl isourea and phosphorylguanidine according to MALDI-TOF MS (Example 7). The latter is likelyformed through substitution of the methoxy group of phosphoryl isoureaby ammonia. Additional ammonia treatment at 55° C. for 16 h resulted inthe increase of the amount of phosphoryl guanidine and the decrease ofthe amount of O-methyl phosphoryl isourea. Likewise, treatment of themixture with ethylenediamine in ethanol (1:1 v/v) at 55° C. for 16 hresulted in disappearance of O-methyl phosphoryl isourea and affordedN-β-aminoethyl phosphoryl guanidine as the major product (Example 8).This opens the way to substituting positively charged groups fornegatively charged phosphates in oligonucleotide sequences as theN-β-aminoethyl phosphoryl guanidine group should be positively chargedat physiological pH. Cationic oligonucleoside phosphoryl guanidines maypotentially exhibit improved cell uptake and in vivo delivery in theabsence of external transfection agents.

As described herein, the present inventors have obtained 20-meroligothymidylates with a single unsubstituted phosphoryl guanidine group(FIG. 2). The yields decreased with the length of oligonucleotide in arow d(T₁₈TpT)>d(T₁₀TpT₉)>d(TTpT₁₈). Oligonucleotides with two and threephosphoryl guanidine groups were also prepared; however, the reactionsresulted in mixtures of products that were difficult to separate,leading the inventors to conclude that iodine oxidation chemistry ismost effective for the preparation of monosubstituted oligonucleotides.

H-Phosphonate chemistry can be used to prepare oligonucleotides with twoand three modifications [63], as described herein. CPG-supported3′,5′-dithymidine-H-phosphonate can be oxidised intoN,N,N′,N′-tetramethyl phosphoryl guanidine either by 0.1 M iodine and20% vol. TMG in pyridine with or without BSA (Examples 1.2 and 1.3) orby CCl₄ or CCl₃Br and 20% vol. TMG in pyridine (Examples 1.4 and 1.5)[64]. The conversion in the case of iodine (70-75%) was higher than inthe case of CCl₄ or CCl₃Br (10-20%). In the presence of BSA theconversion increased to 80-85%. After solid-phase synthesis using thephosphoramidite method, a hexathymidylate 5′-d(TTTTTpT) with tetramethylphosphoryl guanidine group was obtained in good yield together withbyproducts dT₅ and dT₆ (Example 1.3). The inventors have successfullyobtained di- and trisubstituted phosphoryl guanidines 5′-d(TTTTpTpT)(Example 3.1) and 5′-d(TTTpTpTpT) (Example 4.1) via iodine/TMG/BSAoxidation, but in the latter case the yield was lower.

To increase the yield of oligonucleoside phosphoryl guanidines, theinventors explored a novel reaction between CPG-bound dinucleosideβ-cyanoethyl phosphite and tetraalkyl azidocarbenium salts inN,N-dimethylformamide or acetonitrile with or without BSA. The reactionmay occur at ambient temperature or at 40-45° C. Addition of 5%triethylamine as well as BSA increases yield. The method proved to beeffective for the preparation of oligonucleotides with multipletetraalkyl phosphoryl guanidino groups (FIG. 1). An automated version ofthe method on a DNA synthesiser was used to produce fully modifiedoligonucleoside phosphoryl guanidines (Example 47).

Along with charge neutral or cationic phosphoryl guanidines, theinventors have also prepared oligonucleotides containing an ionisableN-cyanoimino phosphate group. In that case, a CPG-bound dinucleosidephosphite was reacted with 0.25 M solution of cyanogen azide inacetonitrile at ambient temperature. The results suggest that the yieldof the oligonucleotide depends on the deprotection method used. Goodresults were obtained when a mixture of ethylenediamine and ethanol (1:1v/v) [65] was substituted for aqueous ammonia and used to deprotect theoligonucleotide with N-cyanoimino phosphate group for 1 h at 70° C.(Examples 40 and 41).

Electrophoretic mobility of the obtained N-cyanoimino hexathymidylates5′-d(TTTTTpT) and 5′-d(TpTTTTT) was very similar to dT₆, which suggeststhat N-cyanoimino group has negative charge at physiological pH. Theoligonucleotides were only slightly hydrophobic than the unmodifiedhexathymidylate. Two diastereomers were observed on RP-HPLC trace (FIG.5).

A range of modified oligonucleotides were found to be compatible withphosphoryl guanidine modifications, in particular ribonucleotidederivatives such as oligo-2′-O-methylribonucleotides (FIG. 4), LNA (FIG.3) and RNA itself (Example 46). Phosphorothioate groups can besuccessfully incorporated into oligonucleotides together with phosphorylguanidine groups (Examples 32-35). A range of other modifications suchas fluorescein, abasic site, nonnucleosidic insert or BlackHole quencherwere tolerated. Mononucleotides with either 3′- or 5′-phosphorylguanidine groups were also prepared (Examples 42 and 44, FIG. 6).

Definitions

The term “nucleotide” refers to a compound containing a nucleoside or amodified nucleoside and at least one phosphate group or a modifiedphosphate group linked to it by a covalent bond. Exemplary covalentbonds include, without limitation, an ester bond between the 3′, 2′ or5′ hydroxyl group of a nucleoside and a phosphate group.

The term “oligonucleotide” refers to a compound containing two or morenucleotides joined together in a polymeric chain. Oligonucleotides maybe deoxyribonucleic acids or ribonucleic acids. Oligonucleotides may besingle stranded or double stranded. In the case of double strandedoligonucleotides one or both strands may contain a modified phosphateaccording to the present invention.

An oligonucleotide may be a polymer of two or more nucleotides, but mayhave any length. For example, an oligonucleotide may have a minimumlength of one of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, or 40 nucleotides. Optionally it may have a maximumlength of one of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200,210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340,350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480,490, or 500 nucleotides, although longer oligonucleotides are alsoprovided in some embodiments. Purely, by way of example anoligonucleotide having one of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100nucleotides is provided.

In oligonucleotides of the present invention one, several (e.g. 2, 3, 4,5, 6, 7, 8, 9, 10 or more) or each nucleotide may contain a modifiedphosphate according to the present invention.

The nucleotides and oligonucleotides of the present invention mayinclude chemical modifications as described herein such as a chemicalsubstitution at a sugar position, a phosphate position, and/or a baseposition of the nucleic acid including, for example, incorporation of amodified nucleotide, incorporation of a capping moiety (e.g. 3′capping), conjugation to a high molecular weight, non-immunogeniccompound (e.g. polyethylene glycol (PEG)), conjugation to a lowmolecular weight compound (e.g. cholesterol), conjugation to a peptide(e.g. a cell-penetrating peptide), substitutions in the phosphate group(e.g. phosphorothioate). Base modifications may include 5-positionpyrimidine modifications, modifications at exocyclic amines,substitution of 4-thiouridine, substitution of 5-bromo- or5-iodo-uracil, backbone modifications. Sugar modifications may include2′-amino nucleotides (2′-NH₂), 2′-fluoro nucleotides (2′-F), 2′-O-methyl(2′-OMe) nucleotides, 2′-O-allyl nucleotides, 2′-O-β-methoxyethylnucleotides, “locked” nucleotides (e.g. LNA) or tricyclo-DNAnucleotides. The bonds between the central phosphorus atom of aphosphate and the or each nucleoside are suitably via oxygen, that is,the 3′ and or 5′ end of the nucleoside is an alcohol. However,nucleoside analogues in which the 3′ and or 5′ end of the nucleoside isnot an alcohol, but rather a suitable analogue, are also envisaged. Forexample, the 3′ and/or 5′ end of a nucleoside may be a thiol, a selenol,or an amine.

Nucleotides and oligonucleotides according to the present invention maybe provided in isolated or purified form.

The term “nucleoside” refers to a compound containing a sugar part and anucleobase. Exemplary sugars include, without limitation, ribose,2-deoxyribose, arabinose and the like. Exemplary nucleobases include,without limitation, thymine, uracil, cytosine, adenine, guanine, purine,hypoxanthine, xanthine, 2-aminopurine, 2,6-diaminopurine,5-methylcytosine, 4-fluorouracil, 5-chlorouracil, 5-bromouracil,5-iodouracil, 5-trifluoromethyluracil, 5-fluorocytosine,5-chlorocytosine, 5-bromocytosine, 5-iodocytosine, 5-propynyluracil,5-propynylcytosine, 7-deazaadenine, 7-deazaguanine,7-deaza-8-azaadenine, 7-deaza-8-azaguanine, isocytosine, isoguanine andthe like.

The term “nucleoside analogues” as used herein refers to a modifiednucleoside in which the sugar part is replaced with any other cyclic oracyclic structure. Exemplary nucleoside analogues in which the sugarpart is replaced with another cyclic structure include, withoutlimitation, monomeric units of morpholinos (PMO) and tricyclo-DNA.Exemplary nucleoside analogues in which the sugar part is replaced withan acyclic structure include, without limitation, monomeric units ofpeptide nucleic acids (PNA) and glycerol nucleic acids (GNA).

The term “nucleoside analogue” additionally refers to a nucleoside anypart of which is replaced by a chemical group of any nature. Exemplarysuch nucleosides analogues include, without limitation, 2′-substitutednucleosides such as 2′-fluoro, 2-deoxy, 2′-O-methyl,2′-O-β-methoxyethyl, 2′-O-allylriboribonucleosides, 2′-amino, lockednucleic acid (LNA) monomers and the like.

Suitably, nucleoside analogues may include nucleoside analogues in whichthe sugar part is replaced by a morpholine ring as depicted below.

In structures of this type, it will be appreciated that the labels 3′and 5′, as applied to conventional sugar chemistry, apply by analogy.That is, in the structure depicted, the hydroxylmethyl substituent onthe ring is considered the 5′ end, while the third nitrogen valency isconsidered the 3′ end.

The term “oligonucleotide analogue” as used herein refers to a modifiedoligonucleotide, which is chemically modified at either its phosphategroups or has its nucleosides replaced by nucleoside analogues.Exemplary oligonucleotide analogues include, without limitation,phosphorothioate oligonucleotides (PS), phosphorodiamidate morpholinooligonucleotides (morpholinos, PMO), tricyclo-DNA and peptide nucleicacids (PNA).

The term “peptide nucleic acid” relates usually to oligonucleotideanalogues that substitute peptide bond for phosphate group. However, asused herein, it includes compounds that may incorporate modifiedphosphate groups according to the present invention. It is understoodthat those compounds would also be covered by the present application.

The term “phosphate group” as used herein refers to phosphoric acidH₃PO₄ wherein any hydrogen atoms are replaced by one, two or threeorganic radicals to give a phosphoester, phosphodiester, orphosphotriester, respectively.

The term “modified phosphate group” refers to a phosphate group whereinany oxygens connected to the phosphorus atom are replaced by a chemicalgroup of any nature. Suitable replacements may include sulfur, selenium,imino (NR) and borane (—BH₃ ⁻). For example, the group may be aphosphorothioate group, a phosphoroselenoate or boranophosphate group.Preferably, the “modified phosphate group” is a phosphorothioate group.

It will be appreciated that, depending on their substitution, thephosphates and modified phosphates described herein may be chiral. Wherestereochemistry is not indicated, the structure encompasses both Rp andSp configurations, each in isolation and as mixtures thereof (forexample, a racemic mixture). For example, and not by way of limitation,the structure:

as depicted encompasses

It will be appreciated that compounds described herein may contain morethan one chiral centre. Except where indicated otherwise, it is intendedthat all enantiomers and diastereomers are encompassed.

The term “protected oligonucleotide” as used herein refers to anoligonucleotide, which incorporates one or more protecting groups.

The term “deprotected oligonucleotide” as used herein refers to anoligonucleotide from which one or more protecting groups have beenremoved.

It will be understood that references to nucleosides, nucleotides, andoligonucleotides include protected forms thereof.

The term “protecting group” refers to a chemical group that is used toblock temporarily a reactive site in a compound. A protecting group isremoved under specific conditions. Exemplary protecting groups include,without limitation, acetyl (Ac), benzoyl (Bz), isobutyryl (Ibu),tert-butylphenoxyacetyl (Tac), levulinyl (Lev), methyl (Me),2-cyanoethyl (CE), allyl (All), 2-chlorophenyl (o-ClPh),4,4′-dimethoxytrityl (DMTr), 4-methoxytrityl (MMTr),tert-butyldimethylsilyl (TBDMS), triisopropylsilyloxymethyl (TOM) andthe like.

The term “linker” as used herein encompasses a chemical group thatconnects a compound to a solid support and is cleavable under specificconditions releasing said compound from said solid support. Exemplarylinkers used in solid-phase oligonucleotide synthesis include, withoutlimitation, succinyl, diglycolyl, oxalyl, hydroquinone-O,O′-diacetyl(Q-linker), phthaloyl, 4,5-dichlorophthaloyl, malonyl, glutaryl,diisopropylsilyl, 1,1,3,3-tetraisopropyldisiloxane-1,3-diyl and thelike.

Other linkers may include non-nucleotide chemical groups inserted intoan oligonucleotide or modified oligonucleotide(“internucleoside/internucleotide linkers”), or non-nucleotide chemicalgroups forming a link between a nucleotide and another chemical moiety,for example a label or quencher. Suitable linkers are known in the artand include 1,12-dodecanediol phosphate (DD).

The term “solid support” refers to a polymeric support used insolid-phase oligonucleotide synthesis. Exemplary solid supports include,without limitation, controlled pore glass (CPG), polystyrene resin,TentaGel® resin, TSK Gel® Toyopearl® resin, poly(vinyl alcohol) resinand the like. The term “solid support” as used herein also refers tonon-resin types of solid supports used, for example, in multipleoligonucleotide synthesis including, without limitation, filter discs,multipin systems, multiwell plates and the like.

The term “organic radical” as used herein refers to a chemical group,which contains one or more carbon atoms connected to any other atoms,with a free valency at carbon. Examples of other atoms include, withoutlimitation, hydrogen, nitrogen, oxygen, fluorine, silicon, phosphorus,sulphur, chlorine, bromine and iodine.

The term alkyl as used herein refers to both straight and branched chaincyclic and acyclic forms. The term “alkyl” includes monovalent, straightor branched, saturated, acyclic hydrocarbyl groups. C₁₋₄alkyl groupsinclude, without limitation, methyl, ethyl, n-propyl, i-propyl ort-butyl groups.

The term “alkenyl” includes monovalent, straight or branched,unsaturated, acyclic hydrocarbyl groups having at least onecarbon-carbon double bond and, in some embodiments, no carbon-carbontriple bonds. In some embodiments alkenyl is C₂₋₁₀alkenyl, in someembodiments C₂₋₆alkenyl, in some embodiments C₂₋₄alkenyl.

The term “alkynyl” includes monovalent, straight or branched,unsaturated, acyclic hydrocarbyl groups having at least onecarbon-carbon triple bond and, in some embodiments, no carbon-carbondouble bonds. In some embodiments, alkynyl is C₂₋₁₀alkynyl, in someembodiments C₂₋₆alkynyl, in some embodiments C₂₋₄alkynyl.

The term “heterocyclic compound” refers to a compound comprising aheterocyclic group. The term “heterocyclic group” refers to group asaturated, partially unsaturated or unsaturated (e.g. aromatic)monocyclic or bicyclic group containing one or more (for example 1, 2,3, 4 or 5) ring heteroatoms selected from O, S(O)_(t) (wherein t is 0,1, or 2) or N and includes unsubstituted groups and groups substitutedwith one or more substituents (for example 1, 2, 3, 4 or 5substituents), optionally wherein the one or more substituents are takentogether to form a further ring system. Unless stated otherwise herein,where a heterocyclic group is bonded to another group, the heterocyclicgroup may be C-linked or N-linked, i.e. it may be linked to theremainder of the molecule through a ring carbon atom or through a ringnitrogen atom (i.e. an endocyclic nitrogen atom). The term heterocyclicgroup thus includes optionally substituted heterocycloalkyl,heterocycloalkenyl and heteroaryl groups as defined herein.

The term “aryl” includes monovalent, aromatic, cyclic hydrocarbylgroups, such as phenyl or naphthyl (e.g. 1-naphthyl or 2-naphthyl). Ingeneral, the aryl groups may be monocyclic or polycyclic fused ringaromatic groups.

The term “heteroaryl” includes aryl groups in which one or more carbonatoms are each replaced by heteroatoms independently selected from O, S,N and NR^(N), where R^(N) is defined herein.

The term “halogen” refers to —F, —Cl, —Br, and —I. In some embodiments,the halogen is —F, —Cl, or —Br. In some embodiments, the halogen is —For —Cl, for example, Cl.

In general, the heteroaryl groups may be monocyclic or polycyclic (e.g.bicyclic) fused ring heteroaromatic groups. Typically, heteroaryl groupscontain 5-10 members wherein 1, 2, 3 or 4 ring members are independentlyselected from O, S, N and NR^(N).

As used herein, the term “optionally substituted” refers to asubstituent that may be substituted with one or more (up to the maximumnumber of free valencies on that substituent) substituents. Thesubstituents may be selected from:

-   -   C₁₋₄alkyl,    -   —F, —Cl, —Br, —I    -   —CF₃, —OCF₃, —SCF₃,    -   —OH, -L-OH, —O-L-OH, —NH-L-OH, —NR³⁰-L-OH,    -   —NR³⁰-L-O    -   —SH,    -   —CN,    -   —NH₂, —NHC₁₋₄alkyl, —N(C₁₋₄alkyl)₂,    -   -L-NH₂, -L-NHC₁₋₄alkyl,    -   —OC(O)C₁₋₄alkyl,    -   —C(O)OH, —C(O)OC₁₋₄alkyl,    -   —C(O)C₁₋₄alkyl,    -   —C(O)NH₂, —C(O)NHC₁₋₄alkyl, —C(O)N(C₁₋₄alkyl)₂,    -   —NHC(O)C₁₋₄alkyl, —N(C₁₋₄alkyl)C(O) C₁₋₄alkyl; and    -   ═O;        wherein each -L- is a bond or a C₁₋₄ alkylene and R³⁰ is —C₁₋₁₀        alkyl, —C₂₋₁₀alkenyl, —C₂₋₁₀alkynyl, —C₆₋₁₀aryl, or        —O₅₋₁₀heteroaryl.

In some embodiments, these optional substituents are selected from—C₁₋₄alkyl, —F, —Cl, —CF₃, —OH, —OC₁₋₄alkyl, —NH₂, —NHC₁₋₄alkyl, and—N(C₁₋₄alkyl)₂.

The term “ambient temperature” as used herein refers to temperatures inthe range of 15-29° C., preferably in the range of 20-25° C.

Reactions

As described herein, a particular advantage of the modified phosphatemoieties of the present invention is the convenient incorporation ofthese moieties as demonstrated by the present inventors. For example, amodified phosphate moiety of Formula VI can be readily incorporated intoan oligonucleotide during sequential oligonucleotide synthesis based onH-phosphonate or phosphoramidite chemistry. Accordingly, the presentinvention provides methods of synthesising the compounds of the presentinvention. These methods may use solid-supported reagents, and may beperformed in a DNA synthesizer.

It will be appreciated that phosphoryl isoureas, phosphorylisothioureas, phosphoryl imidates and phosphoryl imidothioates are,inter alia, reactive intermediates, which upon reaction with primary orsecondary amines can be converted into phosphoryl guanidines orphosphoryl amidines as described herein (see, for example, Examples 7and 8). Accordingly, the present invention also provides a method ofsynthesizing a compound as described herein having a phosphorylguanidine or phosphoryl amidine group, the method comprising reactioninga compound as described herein having a phosphoryl isourea, phosphorylisothiourea, phosphoryl imidate or phosphoryl imidothioate group with aprimary or secondary amine.

H-Phosphonate Chemistry

H-Phosphonate chemistry is a convenient and established method ofchemical oligonucleotide synthesis. Suitably, a 5′-DMT-protectednucleotide affixed via a linker to a solid support undergoes sequentialdeprotection then coupling reaction with an H-phosphonate monoester toform an H-phosphonate diester. Further nucleoside units may be added insequence, following the steps of detritylation then coupling, withoxidation of the internucleosidic H-phosphonate diester linkages tophosphodiester linkages occurring at the end of the assembly with anoxidant, typically iodine.

The present inventors have shown, as described herein, that the modifiedphosphate groups of the invention can be readily incorporated intooligonucleotides assembled using H-phosphonate chemistry. To incorporatea modified phosphate, as described herein, the oxidation may beperformed in the presence of a suitable guanidine, amidine, isourea,isothiourea, imidate or imidothioate which may be present as a free baseor in its salt form, for example, as a hydrochloride or other salt, witha suitable oxidant. Suitable oxidants include iodine I₂, bromine Br₂,chlorine Cl₂, iodine chloride ICI, N-bromosuccinimide,N-chlorosuccinimide, N-iodosuccinimide, carbon tetrachloride CCl₄,bromotrichloromethane CCl₃Br, tetrabromomethane CBr₄, tetraiodomethaneCl₄, iodoform CHI₃, hexachloroethane C₂Cl₆, and hexachloroacetone(CCl₃)₂CO. A preferred oxidant is iodine.

Of course, it will be appreciated that after oxidation to incorporateone or more modified phosphate groups, the cycle of detritylationfollowed by coupling can resume, with further oxidation steps performedat appropriate points during chain assembly. In this way, modifiedphosphate groups according to the invention can be incorporated atappropriate points during the chain assembly.

A variety of solvents known in the art may be used including pyridine,2-picoline, 3-picoline, 4-picoline, quinoline, tetrahydrofuran (THF),1,4-dioxane, 1,2-dimethoxyethane (DME), acetonitrile. Pyridine is apreferred solvent.

It may be preferable to include a base during the oxidation step.Suitably, the base is an amine base, for example, triethylamine,N,N-diisopropylethylamine (DIEA), N-methylmorpholine, N-ethylmorpholine,tributylamine, 1,4-diazabicyclo[2.2.2]octane (DABCO), N-methylimidazole(NMI), pyridine, 2,6-lutidine, 2,4,6-collidine, 4-dimethylaminopyridine(DMAP), 1,8-bis(dimethylamino)naphthalene (“proton sponge”),1,8-diazabicyclo[5.4.0]undec-7-ene (DBU),1,5-diazabicyclo[4.3.0]non-5-ene (DBN),7-methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene (MTBD),2-tert-butyl-1,1,3,3-tetramethylguanidine,2,8,9-trimethyl-2,5,8,9-tetraaza-1-phosphabicyclo[3.3.3]undecane orphosphazene base. For example, the base may be triethylamine or DBU.

The present inventors have found that addition of a silylating reagentduring the oxidation step incorporating the modified phosphate motif(s)may be desirable. Accordingly, N,O-bis(trimethylsilyl)acetamide (BSA),N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA),chlorotrimethylsilane, bromotrimethylsilane, iodotrimethylsilane,triethylsilyl chloride, triphenylsilyl chloride, hexamethyldisilazane,trimethylsilyl trifluoromethanesulfonate (TMSOTf),dimethylisopropylsilyl chloride, diethylisopropylsilyl chloride,tert-butyldimethylsilyl chloride, tert-butyldiphenylsilyl chloride,triisopropylsilyl chloride, dimethyldichlorosilane,diphenyldichlorosilane or similar may be added. A preferred silylatingreagent is N,O-bis(trimethylsilyl)acetamide (BSA).

Phosphoramidite Chemistry

Phosphoramidite chemistry is also an attractive method of chemicaloligonucleotide synthesis. The cycle associated with phosphoramiditechemistry-based oligonucleotide chemistry is known in the art. In brief,a solid supported nucleotide is detritylated, then coupled with asuitably activated nucleoside phosphoramidite to form a phosphitetriester linkage. Capping may then occur, followed by oxidation of thephosphite triester with an oxidant, typically iodine. The cycle is thenrepeated to assemble the chain.

The present inventors have found, as described herein, that performingsuch an oxidation step in the presence of a suitable guanidine, amidine,isourea or isothiourea, which may be present in its salt form, forexample, as a hydrochloride salt, can be used to produce the desiredmodified phosphate group(s). Removal of the β-cyanoethyl phosphiteprotecting group completes the synthesis. As described above for theoxidation step in the H-phosphonate method, the inclusion of basesand/or silylating agents may be desirable.

Use of Organic Azides

Also described herein is a novel reaction to obtain modified phosphategroups of the invention though reaction of a dinucleoside β-cyanoethylphosphite and a suitable organic azide, optionally in the presence of asilylating agent such as BSA. A base, for example an amine base, forexample triethylamine, may be included.

The present inventors have shown that this method is effective for thepreparation of oligonucleotides with various phosphoryl guanidinesubstitution pattern such as having unsubstituted, mono-, di-, tri-, andtetra-substituted phosphoryl guanidine groups, and for oligonucleotideshaving multiple modified phosphate groups of this type.

Suitably, the organic azide can be an azide of formula R¹—C⁺(N₃)—R² suchas a bis(disubstituted amino)-1-azidocarbenium salt, a 1-(disubstitutedamino)-1-azidocarbamidinium salt and the like, or a 1-(disubstitutedamino)-1-azido-ethene, an N-substituted-1-azidocarbamidine; or an azideof formula is (N₃)₂C═NR², (N₃)₂C═N⁺R^(1A)R^(1B), or cyanogen azideN₃—CN. The organic azide may be prepared using methods known in the art.For example, tetraalkyl ureas can be converted toazidobis(dialkylamino)carbenium salts [60], dialkyl amides can beconverted to N,N-dialkyl-azidocarbamidinium salts [61] whilst trialkylureas or monoalkyl amides can afford neutral azides [62].

Suitably, the organic azide may be selected from:

wherein R¹, R², R^(1B), R^(2A), and R^(2B) are as defined herein. Insome embodiments, R² may additionally be —N₃.

For example, the azide may be selected from:

wherein each R^(1A), R^(1B), R^(2A), R^(2B) is independently —H oroptionally substituted C₁₋₁₀ alkyl, —C₂₋₁₀alkenyl, —C₂₋₁₀alkynyl,—C₆₋₁₀aryl, or —C₅₋₁₀heteroaryl; and/or

optionally wherein R^(1A) and R^(2A) together form an alkylene orheteroalkylene chain of 2-4 atoms in length; optionally wherein R^(1A)and R^(2A) together form —CH₂—CH₂— and R^(1B) and R^(2B) are eachindependently selected from —H and methyl.

optionally wherein R^(1A) and R^(1B), together with the atom to whichthey are bound, form a 5-8 membered heterocycle; or

optionally wherein R^(2A) and R^(2B), together with the atom to whichthey are bound, form a 5-8 membered heterocycle, optionally apyrrolidine;

or

wherein each R^(1A) and R^(1B) is independently —H or optionallysubstituted C₁₋₁₀ alkyl, —C₂₋₁₀alkenyl, —C₂₋₁₀alkynyl, —C₆₋₁₀aryl, or—C₆₋₁₀heteroaryl; or R^(1A) and R^(1B), together with the atom to whichthey are bound, form a 5-8 membered heterocycle, and R² is selected from—H, —F, —OPG, —Cl, —Br, —I, —CN, —N₃, —SPG, and —S—C₁₋₁₀alkyl, whereinPG is a protecting group.

In some preferred reactions, the organic azide is:

with a suitable counterion, for example, chloride.

Suitable counterions include, without limitation, chloride Cl⁻, bromideBr⁻, iodide I⁻, triflate (trifluoromethanesulfonate) CF₃SO₃ ⁻,p-toluenesulphonate C₇H₇SO₃ ⁻, dichlorophosphate PO₂Cl₂ ⁻, perchlorateClO₄ ⁻, tetrafluoroborate BF₄ ⁻, tetraphenylborate BPh₄ ⁻,hexafluorophosphate PF₆ ⁻ and the like.

In some embodiments, each of R^(1A), R^(1B), R^(2A), and R^(2B) isindependently —H or optionally substituted C₁₋₁₀alkyl, for example, H oroptionally substituted C₁₋₄alkyl, for example, —H or methyl. In someembodiments, each of R^(1A), R^(1B), R^(2A) and R^(2B) is methyl; thatis, the resultant phosphate is modified with a tetramethyl guanidine.

In some embodiments, R^(1A) and R^(2A) together form an alkylene orheteroalkylene chain of 2-4 atoms in length and R^(1B) and R^(2B) areeach independently selected from —H and —C₁₋₄alkyl. In some embodiments,R^(1A) and R^(2A) together form —CH₂—CH₂—. In some embodiments, R^(1A)and R^(2A) together form —CH₂—CH₂— and R^(1B) and R^(2B) are —H ormethyl.

In some embodiments, R^(1A) and R^(1B), together with the atom to whichthey are bound, form a 5-8 membered heterocycle, preferably pyrrolidine,piperidine, piperazine or morpholine. For example they may, togetherwith the atom to which they are bound, form a 5-membered heterocycle,preferably a pyrrolidine.

In some embodiments, R^(2A) and R^(2B), together with the atom to whichthey are bound, form a 5-8 membered heterocycle, preferably pyrrolidine,piperidine, piperazine or morpholine. For example they may, togetherwith the atom to which they are bound, form a 5-membered heterocycle,preferably a pyrrolidine.

Procedure a and Procedure B

The compounds described herein may be synthesized using Procedure A orProcedure B, as described herein. These Procedures may be used inautomated oligonucleotide synthesis as described herein and illustratedin the appended examples.

Procedure A comprises:

-   -   (i) immersing a solid support to which a protected        oligonucleotide or a protected modified oligonucleotide        containing said phosphorous acid derivative is attached, in a        mixture containing said oxidant, an imino derivative, and,        optionally, a silylating reagent, a base and a solvent;    -   (ii) keeping the solid support immersed whilst maintaining the        temperature within the required range for a period of time        sufficient to ensure the conversion of said phosphorous acid        derivative into a modified phosphate group, thereby producing a        modified oligonucleotide of Formula (I) with a modified        phosphate group;    -   (iii) continuing solid-phase oligonucleotide synthesis according        to desired protocol until the next desired position of        modification, then repeating steps (i) and (ii), or until the        end of the oligonucleotide sequence;    -   (iv) performing desired deprotection and/or cleavage from solid        support, thereby producing a deprotected modified        oligonucleotide of Formula (I) with one or more modified        phosphate groups.

Procedure B Comprises:

-   -   (i) immersing a solid support to which a protected        oligonucleotide or protected modified oligonucleotide containing        said phosphorous acid derivative is attached, in a mixture        containing said organic azide, a solvent, and, optionally, a        silylating reagent and a base;    -   (ii) repeating steps (ii) to (iv) of Procedure A;

The modified oligonucleotide is recovered at the end of the method.

The methods may be performed at a temperature of −20-150° C., preferablyat 0-100° C., and more preferably at 15-80° C.

Preferably, Procedure A is performed at ambient temperature.

The concentration of an imino derivative in Procedure A is suitably0.005-3 M, more preferably 0.1-1.5 M.

The concentration of azide in Procedure B is suitably 0.005-3 M, andpreferably 0.1-1.5 M.

Optionally, a silylating reagent may be added. Suitable silylatingagents include N,O-bis(trimethylsilyl)acetamide (BSA),N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA),chlorotrimethylsilane, bromotrimethylsilane, iodotrimethylsilane,triethylsilyl chloride, triphenylsilyl chloride, hexamethyldisilazane,trimethylsilyl trifluoromethanesulfonate (TMSOTf),dimethylisopropylsilyl chloride, diethylisopropylsilyl chloride,tert-butyldimethylsilyl chloride, tert-butyldiphenylsilyl chloride,triisopropylsilyl chloride, dimethyldichlorosilane,diphenyldichlorosilane and the like. In some preferred embodiments,N,O-bis(trimethylsilyl)acetamide (BSA) is used as a silylating agent.

Optionally, a base may be added. Suitable bases are amine bases, forexample, triethylamine, N,N-diisopropylethylamine (DIEA),N-methylmorpholine, N-ethylmorpholine, tributylamine,1,4-diazabicyclo[2.2.2]octane (DABCO), N-methylimidazole (NMI),pyridine, 2,6-lutidine, 2,4,6-collidine, 4-dimethylaminopyridine (DMAP),1,8-bis(dimethylamino)naphthalene (“proton sponge”),1,8-diazabicyclo[5.4.0]undec-7-ene (DBU),1,5-diazabicyclo[4.3.0]non-5-ene (DBN),1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD),7-methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene (MTBD),1,1,3,3-tetramethylguanidine (TMG),2-tert-butyl-1,1,3,3-tetramethylguanidine,2,8,9-trimethyl-2,5,8,9-tetraaza-1-phosphabicyclo[3.3.3]undecane,phosphazene base and the like.

A preferred base is triethylamine.

Suitable solvents for Procedure A include pyridine, 2-picoline,3-picoline, 4-picoline, quinoline, tetrahydrofuran (THF), 1,4-dioxane,1,2-dimethoxyethane (DME), diethylene glycol dimethyl ether (diglym),diethyl ether, acetonitrile and the like.

A preferred solvent for Procedure A is pyridine.

Suitable solvents for Procedure B include acetonitrile,N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA),N-methylpyrrolidone (NMP), tetramethyl urea,1,3-dimethylimidazolidin-2-one, sulfolane, hexamethyl phosphortriamide(HMPT), 1,4-dioxane, tetrahydrofuran (THF), acetone, ethyl acetate andthe like.

Preferred solvents for Procedure B include N,N-dimethylformamide;N-methylpyrrolidone and acetonitrile.

Oligonucleotide Applications

Oligonucleotides according to the present invention may be used in awide range of applications. For example, they may be used in vitro, e.g.as research or diagnostic agents, or in vivo, e.g. as therapeutic,diagnostic or research agents.

Accordingly, in one aspect of the present invention a method isprovided, the method comprising contacting, in vitro or in vivo, anoligonucleotide according to the present invention (preferably singlestranded) with an oligonucleotide having a high degree of sequenceidentity (preferably single stranded) and allowing the oligonucleotidesto hybridise, (e.g. to form a double stranded oligonucleotide). Themethod may optionally further comprise the step of detecting and/orquantifying the hybridised oligonucleotides. The method may be a methodof detecting a specific oligonucleotide, e.g. mutant, variant, or SingleNucleotide Polymorphism (SNP) containing oligonucleotide. The method maybe a method of diagnosing the presence of a disease in a patient, e.g.involving detection of an oligonucleotide in a sample of tissue orbodily fluid collected from a patient.

Oligonucleotides according to the present invention may be designed andsynthesised to serve as primers in a method of amplification of nucleicacids, such as in Polymerase Chain Reaction (PCR) methods.

Oligonucleotides according to the present invention may be designed andmanufactured as oligonucleotide microarrays (“DNA chips”) to be used inmethods, where the use of oligonucleotide microarrays is necessary.

Oligonucleotides according to the present invention may also be used todesign and synthesise therapeutic nucleic acids, such as siRNA (Angell &Baulcombe (1997) The EMBO Journal 16, 12:3675-3684; and Voinnet &Baulcombe (1997) Nature 389: pg 553); Fire A. et al., Nature, Vol 391,(1998); Fire (1999) Trends Genet. 15: 358-363, Sharp (2001) Genes Dev.15: 485-490, Hammond et al. (2001) Nature Rev. Genes 2: 1110-1119 andTuschl (2001) Chem. Biochem. 2: 239-245), ribozymes, aptamers(Systematic evolution of ligands by exponential enrichment: RNA ligandsto bacteriophage T4 DNA polymerase. Science. 1990 Aug. 3;249(4968):505-10); WO91/19813), DNAzymes, antisense, PMO, PNA, LNA, orgapmers. Oligonucleotide based therapies are well known in the art totreat a variety of diseases, e.g. including in the treatment of viralinfection or viral mediated disease, cancer, disorders of the eye, suchas age-related macular degeneration, prevention of unwantedneovascularisation, exon-splicing deficiency disorders, such as Duchennemuscular dystrophy, and as anti-cholesterol agents. The design ofoligonucleotide agents known or proposed for such treatments may bemodified to incorporate the modified phosphate(s) described herein.Optionally, therapeutic nucleic acids may be conjugated to a deliveryagent such as a cell penetrating peptide (e.g. as described inWO2009/147368).

Accordingly, in one aspect an oligonucleotide according to the presentinvention is provided for use in a method of medical treatment, or foruse in therapy. In another aspect the use of an oligonucleotideaccording to the present invention in the manufacture of a medicament orpharmaceutical composition for use in the treatment of a disease isprovided. In another aspect a method of treatment of a disease isprovided, the method comprising administering an oligonucleotideaccording to the present invention to a patient in need thereof, therebytreating said disease.

Oligonucleotides according to the present invention may be formulated asa medicament or pharmaceutical composition. A medicament orpharmaceutical composition may comprise an oligonucleotide of thepresent invention, e.g. in purified or isolated form, and apharmaceutically acceptable carrier, diluent, excipient or adjuvant.

Medicaments and pharmaceutical compositions according to aspects of thepresent invention may be formulated for administration by a number ofroutes, including but not limited to, parenteral, intravenous,intra-arterial, intramuscular, oral and nasal. The medicaments andcompositions may be formulated in fluid or solid form. Fluidformulations may be formulated for administration by injection to aselected region of the human or animal body.

Administration is preferably in a “therapeutically effective amount”,this being sufficient to show benefit to the individual. The actualamount administered, and rate and time-course of administration, willdepend on the nature and severity of the disease being treated.Prescription of treatment, e.g. decisions on dosage etc., is within theresponsibility of general practitioners and other medical doctors, andtypically takes account of the disorder to be treated, the condition ofthe individual patient, the site of delivery, the method ofadministration and other factors known to practitioners. Examples of thetechniques and protocols mentioned above can be found in Remington'sPharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams &Wilkins.

Methods according to the present invention may be performed in vitro orin vivo. The term “in vitro” is intended to encompass experiments withmaterials, biological substances, cells and/or tissues in laboratoryconditions or in culture whereas the term “in vivo” is intended toencompass experiments and procedures with intact multi-cellularorganisms.

A subject to be treated may be any animal or human. The subject ispreferably mammalian, more preferably human. The subject may be anon-human mammal, but is more preferably human. The subject may be maleor female. The subject may be a patient.

-   -   ---oOo---

The invention includes the combination of the aspects and preferredfeatures described except where such a combination is clearlyimpermissible or expressly avoided.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

Aspects and embodiments of the present invention will now beillustrated, by way of example, with reference to the accompanyingfigures. Further aspects and embodiments will be apparent to thoseskilled in the art. All documents mentioned in this text areincorporated herein by reference.

Throughout this specification, including the claims which follow, unlessthe context requires otherwise, the word “comprise,” and variations suchas “comprises” and “comprising,” will be understood to imply theinclusion of a stated integer or step or group of integers or steps butnot the exclusion of any other integer or step or group of integers orsteps.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise. Ranges may be expressedherein as from “about” one particular value, and/or to “about” anotherparticular value. When such a range is expressed, another embodimentincludes from the one particular value and/or to the other particularvalue. Similarly, when values are expressed as approximations, by theuse of the antecedent “about,” it will be understood that the particularvalue forms another embodiment.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents substitutedwithout departing from the true spirit and scope of the invention.

In addition, many modifications may be made to adapt a particularsituation, material, composition of matter, process, process step orsteps, to the objective spirit and scope of the present invention. Allsuch modifications are intended to be within the scope of the claimsappended hereto.

The invention will now be illustrated by the following non-limitingexamples, with reference to the corresponding Figures.

EXAMPLES

The following examples are provided by way of illustration and are notintended to limit the invention.

General Methods

Modified oligonucleotides were synthesized on a Biosset automated DNAsynthesizer ASM-800 using either β-cyanoethyl phosphoramidite chemistry[58] or H-phosphonate chemistry [66] on 0.2 μmol scale using standard 12μl columns.

All reactions were carried out in 1.5 ml polypropylene tubes with screwcaps and rubber O-rings. After solid-phase synthesis, polymer supportfrom the column was transferred to a plastic tube and treated with 200μl deblocking solution per 5 mg of support. Either conc (ca. 33%) aqammonia solution (Soln A) or 1:1 vol. mixture of ethylenediamine andabs. ethanol (Soln B) was used as a deblocking solution. Afterdeblocking the supernatant was evaporated in vacuo using a SpeedVacconcentrator and 400 μl of 20 mM triethylammonium acetate, pH 7 wasadded, and support removed by centrifugation.

Modified oligonucleotides were purified by reverse-phased (RP) HPLCeither in DMTr OFF or in DMTR ON mode on an Agilent 1200 serieschromatograph using a gradient of acetonitrile from 0 to 40% in 0.02 Mtriethylammonium acetate, pH 7 for 30 min, flow rate 2 cm³ min⁻¹ on aZorbax SB-C18 (5 μm) column (4.6×150 mm). 5′-Terminal DMTr group wasremoved by 15 min treatment with 100 μl 80% acetic acid followed byneutralisation with 400 μl 20 mM triethylammonium acetate, pH 7.0 andevaporation on a SpeedVac concentrator. Then oligonucleotides wereprecipitated by adding 1 ml 1 M LiClO₄ in acetone, the pellet washed byacetone and dried on air for 20 min. Denaturing gel-electrophoresis in20% polyacrylamide gel (PAGE) was used to check purity ofoligonucleotides with bands visualized by staining with Stains-All.Structures of modified oligonucleotides were confirmed bymatrix-assisted laser desorption ionization—time of flight (MALDI-TOF)mass spectra recorded in either negative or positive ion mode on aBruker Reflex III Autoflex Speed mass spectrometer using3-hydroxypicolinic acid as a matrix.

Preparation of 0.5 M Solution of Bis(Dimethylamino)AzidocarbeniumDichlorophosphate.

To a solution of bis(dimethylamino)chlorocarbenium dichlorophosphate(1.105 g, 4.1 mmol) [67] in dry MeCN (10 ml) powdered and dried NaN₃(1.1 equiv, 288 mg, 4.4 mmol) was added. The suspension was stirred for2 h at ambient temperature, filtered, washed with dry MeCN, evaporatedand dried in vacuo, yielding 1.08 g (95%) of oily product. Sixty ninemilligrams of the product were dissolved in 1 ml of DMF-Et₃N 95:5 (v/v),vigorously shaken for 2 min and the precipitate separated bycentrifugation for 3 min at 14,500 rpm to give 0.5 M solution, which canbe stored at ambient temperature for up to a week.

Preparation of 1 M solution of 2-azido-1,3-dimethylimidazoliniumhexafluorophosphate.

2-Chloro-1,3-dimethylimidazolinium hexafluorophosphate (139 mg) and NaN₃(1.1 equiv, 36 mg) were weighed into a 1.5 ml plastic tube, dryacetonitrile (0.5 ml) was added, and the suspension was shaken for 2 hat 30° C., then the precipitate was separated by centrifugation for 5min at 14,500 rpm. The solution was stored at −18° C.

Preparation of 1 M solution of azidodipyrrolidinocarbeniumhexafluorophosphate.

Chlorodipyrrolidinocarbenium hexafluorophosphate (166 mg) and NaN₃ (1.1equiv, 36 mg) were weighed into a 1.5 ml plastic tube, dry acetonitrile(0.5 ml) was added, and the suspension was shaken for 2 h at 30° C.,then the precipitate was separated by centrifugation for 5 min at 14,500rpm. The solution was stored at −18° C.

Example 1. Preparation of a Modified Oligonucleotide 5′-d(TTTTTpT) withthe N,N,N′,N′-Tetramethyl Phosphoryl Guanidine Group (FVIII); p Here andin the Following Examples Indicates Position of the Modified PhosphateGroup

1.1. Procedure (a) with β-Cyanoethyl Phosphite and Iodine.

A column containing 5 mg of High Load 5′-DMTr-dT CPG support (110 μmolg⁻¹) was placed into a DNA synthesizer, and automated β-cyanoethylphosphoramidite solid-phase DNA synthesis was started on 0.2 μmol scale.Synthesis was interrupted after 5′-detritylation, phosphoramiditecoupling and capping but before oxidation. The column was detached fromsynthesiser, drained on a water pump, rinsed with MeCN, and the supportwith attached phosphite was transferred into a plastic tube.

Soln 1 was prepared by dissolving iodine crystals in dry pyridine to 0.2M concentration (51 mg per ml). Soln 2 was prepared by mixing 124 μlN,N,N′,N′-tetramethylguanidine (TMG), 50 μlN,O-bis(trimethylsilyl)acetamide (BSA) and 325 μl dry pyridine, andadding 3 Å molecular sieves to ca. ¼ vol. Aliquots of 20 μl of Soln 1and 2 were mixed in a plastic tube, 10 μl of BSA were added, and after 1min wait the content was transferred to the tube with polymer. The tubewas vortexed for 30 s, centrifugated at 14,500 rpm for 15 s and shakenfor 5 min at ambient temperature. Supernatant was discarded, the supportwas rinsed with 2×200 μl MeCN, transferred into a synthesiser column,and β-cyanoethyl phosphoramidite solid-phase synthesis was resumed untilthe end of the sequence.

The oligonucleotide was detached from support and protecting groupsremoved by Soln A for 1 h at ambient temperature.

Molecular mass: calc. [M] 1860.39, exp. [M+H] 1860.93, [M−H] 1859.14.

1.2. Procedure (a) with H-phosphonate and Iodine.

A column containing 5 mg of 5′-DMTr-dT CPG support (40 μmol g⁻¹) wasplaced into a DNA synthesizer, and automated solid-phase DNA synthesisby H-phosphonate method was started on 0.2 μmol scale. Synthesis wasinterrupted after 5′-detritylation and H-phosphonate coupling, and thecolumn was detached from synthesiser, drained on a water pump, rinsedwith MeCN, and the support with attached dinucleoside H-phosphonate wastransferred into a plastic tube.

Soln 1 was prepared by dissolving iodine crystals in dry pyridine to 0.2M concentration (51 mg per ml). Soln 2 was prepared by mixing 100 μl TMGand 400 μl dry pyridine (20% vol), and adding 3 Å mole cular sieves toca. ¼ vol. Aliquots of 20 μl of Soln 1 and 2 were added to the tube withsupport, vortexed for 30 s, centrifugated at 14,500 rpm for 15 s andshaken for 30 min at ambient temperature. Supernatant was discarded, thesupport was rinsed with 2×200 μl MeCN, transferred into a synthesisercolumn, and β-cyanoethyl phosphoramidite solid-phase synthesis wasresumed until the end of the sequence.

The oligonucleotide was detached from support and protecting groupsremoved by Soln A for 1 h at ambient temperature.

Molecular mass: calc. [M] 1860.39, exp. [M+H] 1861.17, [M−H] 1857.96.

1.3. Procedure (a) with H-phosphonate, BSA and Iodine.

A column containing 5 mg of 5′-DMTr-dT CPG support (40 μmol g⁻¹) wasplaced into a DNA synthesizer, and automated solid-phase DNA synthesisby H-phosphonate method was started on 0.2 μmol scale. Synthesis wasinterrupted after 5′-detritylation and H-phosphonate coupling, and thecolumn was detached from synthesiser, drained on a water pump, rinsedwith MeCN, and the support with attached dinucleoside H-phosphonate wastransferred into a plastic tube.

Soln 1 was prepared by dissolving iodine crystals in dry pyridine to 0.2M concentration (51 mg per ml). Soln 2 was prepared by mixing 125 μl TMG(2 M), 50 μl BSA (1 M) and 325 μl dry pyridine (20% vol), and adding 3 Åmolecular sieves to ca. ¼ vol. Aliquots of 20 μl of Soln 1 and 2 wereadded to the tube with support, 10 μl BSA were added, and the tube wasvortexed for 30 s, centrifugated at 14,500 rpm for 15 s and shaken for 5min at ambient temperature. Supernatant was discarded, the support wasrinsed with 2×200 μl MeCN, transferred into a synthesiser column, andβ-cyanoethyl phosphoramidite solid-phase synthesis was resumed until theend of the sequence.

The oligonucleotide was detached from support and protecting groupsremoved by Soln A for 1 h at ambient temperature.

Molecular mass: calc. [M] 1860.39, exp. [M+H] 1861.17, [M−H] 1857.96.

1.4. Procedure (a) with H-phosphonate and CCl₄.

A column containing 5 mg of 5′-DMTr-dT CPG support (40 μmol g⁻¹) wasplaced into a DNA synthesizer, and automated solid-phase DNA synthesisby H-phosphonate method was started on 0.2 μmol scale. Synthesis wasinterrupted after 5′-detritylation and H-phosphonate coupling, and thecolumn was detached from synthesiser, drained on a water pump, rinsedwith MeCN, and the support with attached dinucleoside H-phosphonate wastransferred into a plastic tube.

Soln 1 was prepared by keeping CCl₄ over 3 Å molecular sieves for 16 h.Soln 2 was prepared by mixing 100 μl TMG and 400 μl dry pyridine (20%vol), and adding 3 Å molecular sieves to ca. ¼ vol. Aliquots of 20 μl ofSoln 1 and 2 were added to the tube with support, vortexed for 30 s,centrifugated at 14,500 rpm for 15 s and shaken for 30 min at ambienttemperature. Supernatant was discarded, the support was rinsed with2×200 μl MeCN, transferred into a synthesiser column, and β-cyanoethylphosphoramidite solid-phase synthesis was resumed until the end of thesequence.

The oligonucleotide was detached from support and protecting groupsremoved by Soln A for 1 h at ambient temperature.

Molecular mass: calc. [M] 1860.39, exp. [M+H] 1860.78, [M−H] 1858.67.

1.5. Procedure (a) with H-Phosphonate and CCl₃Br.

A column containing 5 mg of 5′-DMTr-dT CPG support (40 μmol g⁻¹) wasplaced into a

DNA synthesizer, and automated solid-phase DNA synthesis byH-phosphonate method was started on 0.2 μmol scale. Synthesis wasinterrupted after 5′-detritylation and H-phosphonate coupling, and thecolumn was detached from synthesiser, drained on a water pump, rinsedwith MeCN, and the support with attached dinucleoside H-phosphonate wastransferred into a plastic tube.

Soln 1 was prepared by keeping CCl₃Br over 3 Å molecular sieves for 16h. Soln 2 was prepared by mixing 100 μl TMG and 400 μl dry pyridine (20%vol), and adding 3 Å molecular sieves to ca. ¼ vol. Aliquots of 20 μl ofSoln 1 and 2 were added to the tube with support, vortexed for 30 s,centrifugated at 14,500 rpm for 15 s and shaken for 1 h at ambienttemperature. Supernatant was discarded, the support was rinsed with2×200 μl MeCN, transferred into a synthesiser column, and β-cyanoethylphosphoramidite solid-phase synthesis was resumed until the end of thesequence.

The oligonucleotide was detached from support and protecting groupsremoved by Soln A for 1 h at ambient temperature.

Molecular mass: calc. [M] 1860.39, exp. [M+H] 1861.13, [M−H] 1859.25.

1.6. Procedure (b) with β-Cyanoethyl Phosphite andBis(Dimethylamino)-1-Azidocarbenium Dichlorophosphate.

A column containing 5 mg of High Load 5′-DMTr-dT CPG support (110 μmolg⁻¹) was placed into a DNA synthesizer, and automated β-cyanoethylphosphoramidite solid-phase DNA synthesis was started on 0.2 μmol scale.Synthesis was interrupted after 5′-detritylation, phosphoramiditecoupling and capping but before oxidation. The column was detached fromsynthesiser, drained on a water pump, rinsed with MeCN, and the supportwith attached phosphite was transferred into a plastic tube.

An aliquot of 40 μl 0.5 M bis(dimethylamino)-1-azidocarbeniumdichlorophosphate was transferred into a plastic tube, 10 μul BSA wasadded and the tube was vortexed for 1 min, centrifugated at 14,500 rpmfor 1 min and left for 30 min at ambient temperature. The content wastransferred into the tube with support, vortexed for 30 s, centrifugatedat 14,500 rpm for 30 s and shaken for 30 min at ambient temperature.Supernatant was discarded, the support was rinsed with 2×200 μl MeCN,transferred into a synthesiser column, and β-cyanoethyl phosphoramiditesolid-phase synthesis was resumed until the end of the sequence.

The oligonucleotide was detached from support and protecting groupsremoved by Soln A for 1 h at ambient temperature.

Molecular mass: calc. [M] 1860.39, exp. [M+H] 1860.35, [M−H] 1857.54.

Example 2. Preparation of a Modified Oligonucleotide 5′-d(TpTTTTT) withthe N,N,N′,N′-Tetramethyl Phosphoryl Guanidine Group (FVIII)

A column containing 5 mg of High Load 5′-DMTr-dT CPG support (110 μmolg⁻¹) was placed into a DNA synthesizer, and automated β-cyanoethylphosphoramidite solid-phase DNA synthesis was started on 0.2 μmol scale.Synthesis was interrupted after four dT incorporations followed by5′-detritylation, phosphoramidite coupling and capping but beforeoxidation. The column was detached from synthesiser, drained on a waterpump, rinsed with MeCN, and the support with attached phosphite wastransferred into a plastic tube.

An aliquot of 40 μl 0.5 M bis(dimethylamino)-1-azidocarbeniumdichlorophosphate was transferred into a plastic tube, 10 μl BSA wasadded and the tube was vortexed for 1 min, centrifugated at 14,500 rpmfor 1 min and left for 30 min at ambient temperature. The content wastransferred into the tube with support, vortexed for 30 s, centrifugatedat 14,500 rpm for 30 s and shaken for 30 min at ambient temperature.Supernatant was discarded, the support was rinsed with 2×200 μl MeCN,transferred into a synthesiser column, and the synthesis was completedby 5′-detritylation.

The oligonucleotide was detached from support and protecting groupsremoved by Soln A for 1 h at ambient temperature.

Molecular mass: calc. [M] 1860.39, exp. [M+H] 1860.34, [M−H] 1858.63.

Example 3. Preparation of a Modified Oligonucleotide 5′-d(TTTTpTpT) withthe N,N,N′,N′-tetramethyl Phosphoryl Guanidine Group (FVIII)

3.1. Procedure (a) with H-Phosphonate, BSA and Iodine.

A column containing 5 mg of 5′-DMTr-dT CPG support (40 μmol g⁻¹) wasplaced into a DNA synthesizer, and automated solid-phase DNA synthesisby H-phosphonate method was started on 0.2 μmol scale. Synthesis wasinterrupted after two dT H-phosphonate incorporations, and the columnwas detached from synthesiser, drained on a water pump, rinsed withMeCN, and the support with attached trinucleoside bis-H-phosphonate wastransferred into a plastic tube.

Soln 1 was prepared by dissolving iodine crystals in dry pyridine to 0.2M concentration (51 mg per ml). Soln 2 was prepared by mixing 125 μl TMG(2 M), 50 μl BSA (1 M) and 325 μl dry pyridine (20% vol), and adding 3 Åmolecular sieves to ca. ¼ vol. Aliquots of 20 μl of Soln 1 and 2 wereadded to the tube with support, 10 μl BSA were added, and the tube wasvortexed for 30 s, centrifugated at 14,500 rpm for 15 s and shaken for10 min at ambient temperature. Supernatant was discarded, the supportwas rinsed with 2×200 μl MeCN, transferred into a synthesiser column,and β-cyanoethyl phosphoramidite solid-phase synthesis was resumed untilthe end of the sequence.

The oligonucleotide was detached from support and protecting groupsremoved by Soln A for 1 h at ambient temperature.

Molecular mass: calc. [M] 1957.55, exp. [M+H] 1958.28, [M−H] 1956.34.

3.2. Procedure (b) with β-cyanoethyl Phosphite andbis(dimethylamino)-1-Azidocarbenium Dichlorophosphate.

A column containing 5 mg of High Load 5′-DMTr-dT CPG support (110 μmolg⁻¹) was placed into a DNA synthesizer, and automated β-cyanoethylphosphoramidite solid-phase DNA synthesis was started on 0.2 umol scale.Synthesis was interrupted after 5′-detritylation, phosphoramiditecoupling and capping but before oxidation. The column was detached fromsynthesiser, drained on a water pump, rinsed with MeCN, and the supportwith attached phosphite was transferred into a plastic tube.

An aliquot of 80 μl 0.5 M bis(dimethylamino)-1-azidocarbeniumdichlorophosphate was transferred into a plastic tube, 20 μl BSA wasadded and the tube was vortexed for 1 min, centrifugated at 14,500 rpmfor 1 min and left for 30 min at ambient temperature. An aliquot of 50μl was transferred into the tube with support, vortexed for 30 s,centrifugated at 14,500 rpm for 30 s and shaken for 30 min at ambienttemperature. Supernatant was discarded, the support was rinsed with2×200 μl MeCN, transferred into a synthesiser column, another dTphosphoramidite was coupled and the synthesis was interrupted beforeoxidation. The column was detached from synthesiser, drained on a waterpump, rinsed with MeCN, and the support with attached phosphite wastransferred into a plastic tube.

An aliquot of 50 μl was transferred into the tube with support, vortexedfor 30 s, centrifugated at 14,500 rpm for 30 s and shaken for 30 min atambient temperature. Supernatant was discarded, the support was rinsedwith 2×200 μl MeCN, transferred into a synthesiser column, and thesynthesis was resumed until the end of the sequence.

The oligonucleotide was detached from support and protecting groupsremoved by Soln A for 1 h at ambient temperature.

Molecular mass: calc. [M] 1957.55, exp. [M+H] 1958.32, [M−H] 1955.44.

Example 4. Preparation of a Modified Oligonucleotide 5′-d(TTTpTpTpT)with the N,N,N′,N′-tetramethyl Phosphoryl Guanidine Group (FVIII)

4.1. Procedure (a) with H-phosphonate, BSA and Iodine.

A column containing 5 mg of 5′-DMTr-dT CPG support (40 μmol g⁻¹) wasplaced into a DNA synthesizer, and automated solid-phase DNA synthesisby H-phosphonate method was started on 0.2 μmol scale. Synthesis wasinterrupted after three dT H-phosphonate incorporations, and the columnwas detached from synthesiser, drained on a water pump, rinsed withMeCN, and the support with attached tetranucleoside tris-H-phosphonatewas transferred into a plastic tube.

Soln 1 was prepared by dissolving iodine crystals in dry pyridine to 0.2M concentration (51 mg per ml). Soln 2 was prepared by mixing 125 μl TMG(2 M), 50 μl BSA (1 M) and 325 μl dry pyridine (20% vol), and adding 3 Åmolecular sieves to ca. ¼ vol. Aliquots of 20 μl of Soln 1 and 2 wereadded to the tube with support, 10 μl BSA were added, and the tube wasvortexed for 30 s, centrifugated at 14,500 rpm for 15 s and shaken for10 min at ambient temperature. Supernatant was discarded, the supportwas rinsed with 2×200 μl MeCN, transferred into a synthesiser column,and β-cyanoethyl phosphoramidite solid-phase synthesis was resumed untilthe end of the sequence.

The oligonucleotide was detached from support and protecting groupsremoved by Soln A for 1 h at ambient temperature.

Molecular mass: calc. [M] 2054.72, exp. [M+H] 2055.49.

4.2. Procedure (b) with β-cyanoethyl Phosphite andbis(dimethylamino)-1-Azidocarbenium Dichlorophosphate.

A column containing 5 mg of High Load 5′-DMTr-dT CPG support (110 μmolg⁻¹) was placed into a DNA synthesizer, and automated β-cyanoethylphosphoramidite solid-phase DNA synthesis was started on 0.2 μmol scale.Synthesis was interrupted after 5′-detritylation, phosphoramiditecoupling and capping but before oxidation. The column was detached fromsynthesiser, drained on a water pump, rinsed with MeCN, and the supportwith attached phosphite was transferred into a plastic tube.

An aliquot of 120 μl 0.5 M bis(dimethylamino)-1-azidocarbeniumdichlorophosphate was transferred into a plastic tube, 30 μl BSA wasadded and the tube was vortexed for 1 min, centrifugated at 14,500 rpmfor 1 min and left for 30 min at ambient temperature. An aliquot of 50μl was transferred into the tube with support, vortexed for 30 s,centrifugated at 14,500 rpm for 30 s and shaken for 30 min at ambienttemperature. Supernatant was discarded, the support was rinsed with2×200 μl MeCN, transferred into a synthesiser column, another dTphosphoramidite was coupled and the synthesis was interrupted beforeoxidation. The column was detached from synthesiser, drained on a waterpump, rinsed with MeCN, and the support with attached phosphite wastransferred into a plastic tube.

Second aliquot of 50 μl was transferred into the tube with support,vortexed for 30 s, centrifugated at 14,500 rpm for 30 s and shaken for30 min at ambient temperature. Supernatant was discarded, the supportwas rinsed with 2×200 μl MeCN, transferred into a synthesiser column,another dT phosphoramidite was coupled and the synthesis was interruptedbefore oxidation. The column was detached from synthesiser, drained on awater pump, rinsed with MeCN, and the support with attached phosphitewas transferred into a plastic tube.

Third aliquot of 50 μl was transferred into the tube with support,vortexed for 30 s, centrifugated at 14,500 rpm for 30 s and shaken for30 min at ambient temperature. Supernatant was discarded, the supportwas rinsed with 2×200 μl MeCN, transferred into a synthesiser column,and the synthesis was resumed until the end of the sequence.

The oligonucleotide was detached from support and protecting groupsremoved by Soln A for 1 h at ambient temperature.

Molecular mass: calc. [M] 2054.72, exp. [M+H] 2054.49.

Example 5. Preparation of a Modified Oligonucleotide 5′-d(TTTTTpT) withthe Phosphoryl Guanidine Group (FIX)

A column containing 5 mg of High Load 5′-DMTr-dT CPG support (110 μmolg⁻¹) was placed into a DNA synthesizer, and automated β-cyanoethylphosphoramidite solid-phase DNA synthesis was started on 0.2 umol scale.Synthesis was interrupted after 5′-detritylation, phosphoramiditecoupling and capping but before oxidation. The column was detached fromsynthesiser, drained on a water pump, rinsed with MeCN, and the supportwith attached phosphite was transferred into a plastic tube.

Soln 1 was prepared by dissolving iodine crystals in dry pyridine to 0.2M concentration (51 mg per ml). Soln 2 was prepared by weighing 9.6 mgof dried guanidine hydrochloride into a plastic tube, adding 85 μl drypyridine, 15 μl 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), 10 μl BSA, andthe tube was vortexed for 5 min and sonicated until clear (ca. 5 min).Aliquots of 20 μl of Soln 1 and 2 were mixed in a plastic tube, 10 μl ofBSA were added, and after 1 min wait the content was transferred to thetube with polymer. The tube was vortexed for 30 s, centrifugated at14,500 rpm for 15 s and shaken for 5 min at ambient temperature.Supernatant was discarded, the support was rinsed with 2×200 μl MeCN,transferred into a synthesiser column, and β-cyanoethyl phosphoramiditesolid-phase synthesis was resumed until the end of the sequence.

The oligonucleotide was detached from support and protecting groupsremoved by Soln A for 1 h at ambient temperature.

Molecular mass: calc. [M] 1804.28, exp. [M+H] 1804.62, [M−H] 1803.25.

Example 6. Preparation of a Modified Oligonucleotide 5′-d(TpTTTTT) withthe Phosphoryl Guanidine Group (FIX)

A column containing 5 mg of High Load 5′-DMTr-dT CPG support (110 μmolg⁻¹) was placed into a DNA synthesizer, and automated β-cyanoethylphosphoramidite solid-phase DNA synthesis was started on 0.2 umol scale.Synthesis was interrupted after four dT incorporations followed by5′-detritylation, phosphoramidite coupling and capping but beforeoxidation. The column was detached from synthesiser, drained on a waterpump, rinsed with MeCN, and the support with attached phosphite wastransferred into a plastic tube.

Soln 1 was prepared by dissolving iodine crystals in dry pyridine to 0.2M concentration (51 mg per ml). Soln 2 was prepared by weighing 9.6 mgof dried guanidine hydrochloride into a plastic tube, adding 85 μl drypyridine, 15 μl DBU, 10 μl BSA, and the tube was vortexed for 5 min andsonicated until clear (ca. 5 min). Aliquots of 20 μl of Soln 1 and 2were mixed in a plastic tube, 10 μl of BSA were added, and after 1 minwait the content was transferred to the tube with polymer. The tube wasvortexed for 30 s, centrifugated at 14,500 rpm for 15 s and shaken for 5min at ambient temperature. Supernatant was discarded, the support wasrinsed with 2×200 μl MeCN, transferred into a synthesiser column, andthe synthesis was completed by 5′-detritylation.

The oligonucleotide was detached from support and protecting groupsremoved by Soln A for 1 h at ambient temperature.

Molecular mass: calc. [M] 1804.28, exp. [M+H] 1804.76, [M−H] 1803.06.

Example 7. Preparation of a Modified Oligonucleotide 5′-d(TTTTTpT) withthe O-methyl Phosphoryl Isourea Group (FX)

A column containing 5 mg of High Load 5′-DMTr-dT CPG support (110 μmolg⁻¹) was placed into a DNA synthesizer, and automated β-cyanoethylphosphoramidite solid-phase DNA synthesis was started on 0.2 μmol scale.Synthesis was interrupted after 5′-detritylation, phosphoramiditecoupling and capping but before oxidation. The column was detached fromsynthesiser, drained on a water pump, rinsed with MeCN, and the supportwith attached phosphite was transferred into a plastic tube.

Soln 1 was prepared by dissolving iodine crystals in dry pyridine to 0.2M concentration (51 mg per ml). Soln 2 was prepared by weighing 8.6 mgof dried O-methylisourea hydrogen sulfate into a plastic tube, adding 35μl dry pyridine, 15 μl 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), 5 μlBSA, and the tube was vortexed for 5 min and sonicated until clear (ca.10 min) followed by centrifugation at 14,500 rpm for 2 min. Aliquots of20 μl of Soln 1 and 2 were mixed in a plastic tube, 10 μl of BSA wereadded, and after 1 min wait the content was transferred to the tube withpolymer. The tube was vortexed for 30 s, centrifugated at 14,500 rpm for15 s and shaken for 5 min at ambient temperature. Supernatant wasdiscarded, the support was rinsed with 2×200 μl MeCN, transferred into asynthesiser column, and the synthesis was resumed until the end of thesequence.

The oligonucleotide was detached from support and protecting groupsremoved by Soln

A for 16 h at 55° C.

Molecular mass: calc. [M] 1819.29, exp. [M+H] 1819.59, [M−H] 1818.26.

Example 8. Preparation of a Modified Oligonucleotide 5′-d(TTTTTpT) withthe N-β-aminoethyl Phosphoryl Guanidine Group (FXI)

A column containing 5 mg of High Load 5′-DMTr-dT CPG support (110 μmolg⁻¹) was placed into a DNA synthesizer, and automated β-cyanoethylphosphoramidite solid-phase DNA synthesis was started on 0.2 umol scale.Synthesis was interrupted after 5′-detritylation, phosphoramiditecoupling and capping but before oxidation. The column was detached fromsynthesiser, drained on a water pump, rinsed with MeCN, and the supportwith attached phosphite was transferred into a plastic tube.

Soln 1 was prepared by dissolving iodine crystals in dry pyridine to 0.2M concentration (51 mg per ml). Soln 2 was prepared by weighing 8.6 mgof dried O-methylisourea hydrogen sulfate into a plastic tube, adding 35μl dry pyridine, 15 μl 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), 5 μlBSA, and the tube was vortexed for 5 min and sonicated until clear (ca.10 min) followed by centrifugation at 14,500 rpm for 2 min. Aliquots of20 μl of Soln 1 and 2 were mixed in a plastic tube, 10 μl of BSA wereadded, and after 1 min wait the content was transferred to the tube withpolymer. The tube was vortexed for 30 s, centrifugated at 14,500 rpm for15 s and shaken for 5 min at ambient temperature. Supernatant wasdiscarded, the support was rinsed with 2×200 μl MeCN, transferred into asynthesiser column, and the synthesis was resumed until the end of thesequence.

The oligonucleotide was detached from support and protecting groupsremoved by Soln B for 16 h at 55° C.

Molecular mass: calc. [M] 1847.35, exp. [M+H] 1847.16, [M−H] 1844.76.

Example 9. Preparation of a Modified Oligonucleotide 5′-d(TCpA) with theN,N,N′,N′-tetramethyl Phosphoryl Guanidine Group (FVIII)

A column containing 5 mg of 5′-DMTr-dA(Bz) CPG support (110 μmol g⁻¹)was placed into a DNA synthesiser, and automated β-cyanoethylphosphoramidite solid-phase DNA synthesis was started on 0.2 μmol scale.Synthesis was interrupted after 5′-detritylation, dC phosphoramiditecoupling and capping but before oxidation. The column was detached fromsynthesiser, drained on a water pump, rinsed with MeCN, and the supportwith attached phosphite was transferred into a plastic tube.

An aliquot of 40 μl 0.5 M bis(dimethylamino)-1-azidocarbeniumdichlorophosphate was transferred into a plastic tube, 10 μl BSA wasadded and the tube was vortexed for 1 min, centrifugated at 14,500 rpmfor 1 min and left for 30 min at ambient temperature. The content wastransferred into the tube with support, vortexed for 30 s, centrifugatedat 14,500 rpm for 30 s and shaken for 30 min at ambient temperature.Supernatant was discarded, the support was rinsed with 2×200 μl MeCN,transferred into a synthesiser column, and the synthesis was resumeduntil the end of the sequence.

The oligonucleotide was detached from support and protecting groupsremoved by Soln A for 16 h at ambient temperature.

Molecular mass: calc. [M] 941.80, exp. [M+H] 942.17, [M−H] 938.97.

³¹P NMR (D₂O, δ, ppm): “fast” diastereomer 0.37, “slow” diastereomer0.21.

Example 10. Preparation of a Modified Oligonucleotide 5′-d(GCGCCAAACpA)(SEQ ID NO: 11) with the N,N,N′,N′-tetramethyl Phosphoryl GuanidineGroup (FVIII)

A column containing 5 mg of 5′-DMTr-dA(Bz) CPG support (110 μmol g⁻¹)was placed into a DNA synthesiser, and automated β-cyanoethylphosphoramidite solid-phase DNA synthesis was started on 0.2 μmol scale.Synthesis was interrupted after 5′-detritylation, dC phosphoramiditecoupling and capping but before oxidation. The column was detached fromsynthesiser, drained on a water pump, rinsed with MeCN, and the supportwith attached phosphite was transferred into a plastic tube.

An aliquot of 40 μl 0.5 M bis(dimethylamino)-1-azidocarbeniumdichlorophosphate was transferred into a plastic tube, 10 μl BSA wasadded and the tube was vortexed for 1 min, centrifugated at 14,500 rpmfor 1 min and left for 30 min at ambient temperature. The content wastransferred into the tube with support, vortexed for 30 s, centrifugatedat 14,500 rpm for 30 s and shaken for 30 min at ambient temperature.Supernatant was discarded, the support was rinsed with 2×200 μl MeCN,transferred into a synthesiser column, and the synthesis was resumeduntil the end of the sequence.

The oligonucleotide was detached from support and protecting groupsremoved by Soln A for 12 h at 55° C. An HPLC trace of theoligonucleotide is given in FIG. 1.

Molecular mass: calc. [M] 3103.21, exp. [M+H] 3102.12, [M−H] 3100.61.

Example 11. Preparation of a Modified Oligonucleotide 5′-d(GCGCCAAApCpA)(SEQ ID NO: 7) with the N,N,N′,N′-tetramethyl Phosphoryl Guanidine Group(FVIII)

A column containing 5 mg of 5′-DMTr-dA(Bz) CPG support (110 μmol g⁻¹)was placed into a DNA synthesiser, and automated β-cyanoethylphosphoramidite solid-phase DNA synthesis was started on 0.2 μmol scale.Synthesis was interrupted after 5′-detritylation, dC phosphoramiditecoupling and capping but before oxidation. The column was detached fromsynthesiser, drained on a water pump, rinsed with MeCN, and the supportwith attached phosphite was transferred into a plastic tube.

An aliquot of 80 μl 0.5 M bis(dimethylamino)-1-azidocarbeniumdichlorophosphate was transferred into a plastic tube, 20 μl BSA wasadded and the tube was vortexed for 1 min, centrifugated at 14,500 rpmfor 1 min and left for 30 min at ambient temperature. An aliquot of 50μl was transferred into the tube with support, vortexed for 30 s,centrifugated at 14,500 rpm for 30 s and shaken for 30 min at ambienttemperature. Supernatant was discarded, the support was rinsed with2×200 μl MeCN, transferred into a synthesiser column, a dAphosphoramidite was coupled and the synthesis was interrupted beforeoxidation. The column was detached from synthesiser, drained on a waterpump, rinsed with MeCN, and the support with attached phosphite wastransferred into a plastic tube.

Second aliquot of 50 μl was transferred into the tube with support,vortexed for 30 s, centrifugated at 14,500 rpm for 30 s and shaken for30 min at ambient temperature. Supernatant was discarded, the supportwas rinsed with 2×200 μl MeCN, transferred into a synthesiser column,and the synthesis was resumed until the end of the sequence.

The oligonucleotide was detached from support and protecting groupsremoved by Soln A for 12 h at 55° C. An HPLC trace of theoligonucleotide is given in FIG. 1.

Molecular mass: calc. [M] 3200.38, exp. [M+H] 3199.90, [M−H] 3199.00.

Example 12. Preparation of a Modified Oligonucleotide 5′-d(GCGCCAApACpA)(SEQ ID NO: 14) with the N,N,N′,N′-tetramethyl Phosphoryl GuanidineGroup (FVIII)

A column containing 5 mg of 5′-DMTr-dA(Bz) CPG support (110 μmol g⁻¹)was placed into a DNA synthesiser, and automated β-cyanoethylphosphoramidite solid-phase DNA synthesis was started on 0.2 μmol scale.Synthesis was interrupted after 5′-detritylation, dC phosphoramiditecoupling and capping but before oxidation. The column was detached fromsynthesiser, drained on a water pump, rinsed with MeCN, and the supportwith attached phosphite was transferred into a plastic tube.

An aliquot of 80 μl 0.5 M bis(dimethylamino)-1-azidocarbeniumdichlorophosphate was transferred into a plastic tube, 20 μl BSA wasadded and the tube was vortexed for 1 min, centrifugated at 14,500 rpmfor 1 min and left for 30 min at ambient temperature. An aliquot of 50μl was transferred into the tube with support, vortexed for 30 s,centrifugated at 14,500 rpm for 30 s and shaken for 30 min at ambienttemperature. Supernatant was discarded, the support was rinsed with2×200 μl MeCN, transferred into a synthesiser column, then a dAphosphoramidite was coupled followed by another dA, and the synthesiswas interrupted before the last oxidation step. The column was detachedfrom synthesiser, drained on a water pump, rinsed with MeCN, and thesupport with attached phosphite was transferred into a plastic tube.

Second aliquot of 50 μl was transferred into the tube with support,vortexed for 30 s, centrifugated at 14,500 rpm for 30 s and shaken for30 min at ambient temperature. Supernatant was discarded, the supportwas rinsed with 2×200 μl MeCN, transferred into a synthesiser column,and the synthesis was resumed until the end of the sequence.

The oligonucleotide was detached from support and protecting groupsremoved by Soln A for 12 h at 55° C.

Molecular mass: calc. [M] 3200.38, exp. [M−H] 3199.51.

Example 13. Preparation of a Modified Oligonucleotide 5′-d(GCGCCApAACpA)(SEQ ID NO: 15) with the N,N,N′,N′-tetramethyl Phosphoryl GuanidineGroup (FVIII)

A column containing 5 mg of 5′-DMTr-dA(Bz) CPG support (110 μmol g⁻¹)was placed into a DNA synthesiser, and automated β-cyanoethylphosphoramidite solid-phase DNA synthesis was started on 0.2 μmol scale.Synthesis was interrupted after 5′-detritylation, dC phosphoramiditecoupling and capping but before oxidation. The column was detached fromsynthesiser, drained on a water pump, rinsed with MeCN, and the supportwith attached phosphite was transferred into a plastic tube.

An aliquot of 80 μl 0.5 M bis(dimethylamino)-1-azidocarbeniumdichlorophosphate was transferred into a plastic tube, 20 μl BSA wasadded and the tube was vortexed for 1 min, centrifugated at 14,500 rpmfor 1 min and left for 30 min at ambient temperature. An aliquot of 50μl was transferred into the tube with support, vortexed for 30 s,centrifugated at 14,500 rpm for 30 s and shaken for 30 min at ambienttemperature. Supernatant was discarded, the support was rinsed with2×200 μl MeCN, transferred into a synthesiser column, then two dAphosphoramidites were coupled followed by another dA, and the synthesiswas interrupted before the last oxidation step. The column was detachedfrom synthesiser, drained on a water pump, rinsed with MeCN, and thesupport with attached phosphite was transferred into a plastic tube.

Second aliquot of 50 μl was transferred into the tube with support,vortexed for 30 s, centrifugated at 14,500 rpm for 30 s and shaken for30 min at ambient temperature. Supernatant was discarded, the supportwas rinsed with 2×200 μl MeCN, transferred into a synthesiser column,and the synthesis was resumed until the end of the sequence.

The oligonucleotide was detached from support and protecting groupsremoved by Soln A for 12 h at 55° C.

Molecular mass: calc. [M] 3200.38, exp. [M−H] 3199.15.

Example 14. Preparation of a Modified Oligonucleotide5′-d(GCGCCAApApCpA) (SEQ ID NO: 12) with the N,N,N′,N′-tetramethylPhosphoryl Guanidine Group (FVIII)

A column containing 5 mg of 5′-DMTr-dA(Bz) CPG support (110 μmol g⁻¹)was placed into a DNA synthesiser, and automated β-cyanoethylphosphoramidite solid-phase DNA synthesis was started on 0.2 μmol scale.Synthesis was interrupted after 5′-detritylation, dC phosphoramiditecoupling and capping but before oxidation. The column was detached fromsynthesiser, drained on a water pump, rinsed with MeCN, and the supportwith attached phosphite was transferred into a plastic tube.

An aliquot of 120 μl 0.5 M bis(dimethylamino)-1-azidocarbeniumdichlorophosphate was transferred into a plastic tube, 30 μl BSA wasadded and the tube was vortexed for 1 min, centrifugated at 14,500 rpmfor 1 min and left for 30 min at ambient temperature. An aliquot of 50μl was transferred into the tube with support, vortexed for 30 s,centrifugated at 14,500 rpm for 30 s and shaken for 30 min at ambienttemperature. Supernatant was discarded, the support was rinsed with2×200 μl MeCN, transferred into a synthesiser column, a dAphosphoramidite was coupled and the synthesis was interrupted beforeoxidation. The column was detached from synthesiser, drained on a waterpump, rinsed with MeCN, and the support with attached phosphite wastransferred into a plastic tube.

Second aliquot of 50 μl was transferred into the tube with support,vortexed for 30 s, centrifugated at 14,500 rpm for 30 s and shaken for30 min at ambient temperature. Supernatant was discarded, the supportwas rinsed with 2×200 μl MeCN, transferred into a synthesiser column, adA phosphoramidite was coupled and the synthesis was interrupted beforeoxidation. The column was detached from synthesiser, drained on a waterpump, rinsed with MeCN, and the support with attached phosphite wastransferred into a plastic tube.

Third aliquot of 50 μl was transferred into the tube with support,vortexed for 30 s, centrifugated at 14,500 rpm for 30 s and shaken for30 min at ambient temperature. Supernatant was discarded, the supportwas rinsed with 2×200 μl MeCN, transferred into a synthesiser column,and the synthesis was resumed until the end of the sequence.

The oligonucleotide was detached from support and protecting groupsremoved by Soln A for 12 h at 55° C. An HPLC trace of theoligonucleotide is given in FIG. 1.

Molecular mass: calc. [M] 3297.54, exp. [M−H] 3296.80.

Example 15. Preparation of a Modified Oligonucleotide5′-d(GGAAGGGGAGAGpA) (SEQ ID NO: 3) with the N,N,N′,N′-tetramethylPhosphoryl Guanidine Group (FVIII)

A column containing 5 mg of 5′-DMTr-dA(Bz) CPG support (110 μmol g⁻¹)was placed into a DNA synthesiser, and automated β-cyanoethylphosphoramidite solid-phase DNA synthesis was started on 0.2 μmol scale.Synthesis was interrupted after 5′-detritylation, dG phosphoramiditecoupling and capping but before oxidation. The column was detached fromsynthesiser, drained on a water pump, rinsed with MeCN, and the supportwith attached phosphite was transferred into a plastic tube.

An aliquot of 40 μl 0.5 M bis(dimethylamino)-1-azidocarbeniumdichlorophosphate was transferred into a plastic tube, 10 μl BSA wasadded and the tube was vortexed for 1 min, centrifugated at 14,500 rpmfor 1 min and left for 30 min at ambient temperature. The content wastransferred into the tube with support, vortexed for 30 s, centrifugatedat 14,500 rpm for 30 s and shaken for 30 min at ambient temperature.Supernatant was discarded, the support was rinsed with 2×200 μl MeCN,transferred into a synthesiser column, and the synthesis was resumeduntil the end of the sequence.

The oligonucleotide was detached from support and protecting groupsremoved by Soln A for 16 h at 55° C.

Molecular mass: calc. [M] 4234.94, exp. [M−H] 4233.84.

Example 16. Preparation of a Modified Oligonucleotide5′-d(TTTTTTTTTTTTTTTTTTTpT) (SEQ ID NO: 16) with theN,N,N′,N′-tetramethyl Phosphoryl Guanidine Group (FVIII)

A column containing 5 mg of High Load 5′-DMTr-dT CPG support (110 μmolg⁻¹) was placed into a DNA synthesizer, and automated β-cyanoethylphosphoramidite solid-phase DNA synthesis was started on 0.2 umol scale.Synthesis was interrupted after 5′-detritylation, phosphoramiditecoupling and capping but before oxidation. The column was detached fromsynthesiser, drained on a water pump, rinsed with MeCN, and the supportwith attached phosphite was transferred into a plastic tube.

Soln 1 was prepared by dissolving iodine crystals in dry pyridine to 0.2M concentration (51 mg per ml). Soln 2 was prepared by mixing 124 μlTMG, 50 μl BSA and 325 μl dry pyridine, and adding 3 Å molecular sievesto ca. ¼ vol. Aliquots of 20 μl of Soln 1 and 2 were mixed in a plastictube, 10 μl of BSA were added, and after 1 min wait the content wastransferred to the tube with polymer. The tube was vortexed for 30 s,centrifugated at 14,500 rpm for 15 s and shaken for 5 min at ambienttemperature. Supernatant was discarded, the support was rinsed with2×200 μl MeCN, transferred into a synthesiser column, and β-cyanoethylphosphoramidite solid-phase synthesis was resumed until the end of thesequence.

The oligonucleotide was detached from support and protecting groupsremoved by Soln A for 2 h at ambient temperature.

Molecular mass: calc. [M] 6119.16, exp. [M+H] 6121.13, [M−H] 6113.80.

Example 17. Preparation of a Modified Oligonucleotide5′-d(TTTTTTTTTTTpTTTTTTTTT) (SEQ ID NO: 17) with theN,N,N′,N′-tetramethyl Phosphoryl Guanidine Group (FVIII)

A column containing 5 mg of High Load 5′-DMTr-dT CPG support (110 μmolg⁻¹) was placed into a DNA synthesizer, and automated β-cyanoethylphosphoramidite solid-phase DNA synthesis was started on 0.2 umol scale.Synthesis was interrupted after eight cycles of dT incorporationfollowed by 5′-detritylation, another dT phosphoramidite coupling andcapping but before oxidation. The column was detached from synthesiser,drained on a water pump, rinsed with MeCN, and the support with attachedphosphite was transferred into a plastic tube.

Soln 1 was prepared by dissolving iodine crystals in dry pyridine to 0.2M concentration (51 mg per ml). Soln 2 was prepared by mixing 124 μlTMG, 50 μl BSA and 325 μl dry pyridine, and adding 3 Å molecular sievesto ca. ¼ vol. Aliquots of 20 μl of Soln 1 and 2 were mixed in a plastictube, 10 μl of BSA were added, and after 1 min wait the content wastransferred to the tube with polymer. The tube was vortexed for 30 s,centrifugated at 14,500 rpm for 15 s and shaken for 5 min at ambienttemperature. Supernatant was discarded, the support was rinsed with2×200 μl MeCN, transferred into a synthesiser column, and β-cyanoethylphosphoramidite solid-phase synthesis was resumed until the end of thesequence.

The oligonucleotide was detached from support and protecting groupsremoved by Soln A for 2 h at ambient temperature.

Molecular mass: calc. [M] 6119.16, exp. [M+H] 6121.54.

Example 18. Preparation of a Modified Oligonucleotide5′-d(TTpTTTTTTTTTTTTTTTTTT) (SEQ ID NO: 18) with theN,N,N′,N′-tetramethyl Phosphoryl Guanidine Group (FVIII)

A column containing 5 mg of High Load 5′-DMTr-dT CPG support (110 μmolg⁻¹) was placed into a DNA synthesizer, and automated β-cyanoethylphosphoramidite solid-phase DNA synthesis was started on 0.2 μmol scale.Synthesis was interrupted after 17 cycles of dT incorporation followedby 5′-detritylation, another dT phosphoramidite coupling and capping butbefore oxidation. The column was detached from synthesiser, drained on awater pump, rinsed with MeCN, and the support with attached phosphitewas transferred into a plastic tube.

Soln 1 was prepared by dissolving iodine crystals in dry pyridine to 0.2M concentration (51 mg per ml). Soln 2 was prepared by mixing 124 μlTMG, 50 μl BSA and 325 μl dry pyridine, and adding 3 Å molecular sievesto ca. ¼ vol. Aliquots of 20 μl of Soln 1 and 2 were mixed in a plastictube, 10 μl of BSA were added, and after 1 min wait the content wastransferred to the tube with polymer. The tube was vortexed for 30 s,centrifugated at 14,500 rpm for 15 s and shaken for 5 min at ambienttemperature. Supernatant was discarded, the support was rinsed with2×200 μl MeCN, transferred into a synthesiser column, and β-cyanoethylphosphoramidite solid-phase synthesis was resumed until the end of thesequence.

The oligonucleotide was detached from support and protecting groupsremoved by Soln A for 2 h at ambient temperature.

Molecular mass: calc. [M] 6119.16, exp. [M+H] 6120.01, [M−H] 6114.19.

Example 19. Preparation of a Modified Oligonucleotide5′-d(TTpTTTTTTTTTTTTTTTTTpT) (SEQ ID NO: 19) with theN,N,N′,N′-tetramethyl Phosphoryl Guanidine Group (FVIII)

A column containing 5 mg of High Load 5′-DMTr-dT CPG support (110 μmolg⁻¹) was placed into a DNA synthesizer, and automated β-cyanoethylphosphoramidite solid-phase DNA synthesis was started on 0.2 umol scale.Synthesis was interrupted after 5′-detritylation, dT phosphoramiditecoupling and capping but before oxidation. The column was detached fromsynthesiser, drained on a water pump, rinsed with MeCN, and the supportwith attached phosphite was transferred into a plastic tube.

An aliquot of 80 μl 0.5 M bis(dimethylamino)-1-azidocarbeniumdichlorophosphate was transferred into a plastic tube, 20 μl BSA wasadded and the tube was vortexed for 1 min, centrifugated at 14,500 rpmfor 1 min and left for 30 min at ambient temperature. An aliquot of 50μl was transferred into the tube with support, vortexed for 30 s,centrifugated at 14,500 rpm for 30 s and shaken for 30 min at ambienttemperature. Supernatant was discarded, the support was rinsed with2×200 μl MeCN, transferred into a synthesiser column, the synthesis wasresumed by incorporating 17 dT nucleotides and stopped after another dTcoupling and capping but before oxidation. The column was detached fromsynthesiser, drained on a water pump, rinsed with MeCN, and the supportwith attached phosphite was transferred into a plastic tube.

Second aliquot of 50 μl was transferred into the tube with support,vortexed for 30 s, centrifugated at 14,500 rpm for 30 s and shaken for30 min at ambient temperature. Supernatant was discarded, the supportwas rinsed with 2×200 μl MeCN, transferred into a synthesiser column,and the synthesis was resumed until the end of the sequence.

The oligonucleotide was detached from support and protecting groupsremoved by Soln A for 2 h at ambient temperature.

Molecular mass: calc. [M] 6216.33, exp. [M−H] 6221.11.

Example 20. Preparation of a Modified Oligonucleotide5′-d(TTpTTTTTTTTTpTTTTTTTTpT) (SEQ ID NO: 20) with theN,N,N′,N′-tetramethyl Phosphoryl Guanidine Group (FVIII)

A column containing 5 mg of High Load 5′-DMTr-dT CPG support (110 μmolg⁻¹) was placed into a DNA synthesiser, and automated β-cyanoethylphosphoramidite solid-phase DNA synthesis was started on 0.2 μmol scale.Synthesis was interrupted after 5′-detritylation, dT phosphoramiditecoupling and capping but before oxidation. The column was detached fromsynthesiser, drained on a water pump, rinsed with MeCN, and the supportwith attached phosphite was transferred into a plastic tube.

An aliquot of 120 μl 0.5 M bis(dimethylamino)-1-azidocarbeniumdichlorophosphate was transferred into a plastic tube, 30 μl BSA wasadded and the tube was vortexed for 1 min, centrifugated at 14,500 rpmfor 1 min and left for 30 min at ambient temperature. An aliquot of 50μl was transferred into the tube with support, vortexed for 30 s,centrifugated at 14,500 rpm for 30 s and shaken for 30 min at ambienttemperature. Supernatant was discarded, the support was rinsed with2×200 μl MeCN, transferred into a synthesiser column, eight dTnucleotides were incorporated and the synthesis was interrupted on thelast dT before oxidation. The column was detached from synthesiser,drained on a water pump, rinsed with MeCN, and the support with attachedphosphite was transferred into a plastic tube.

Second aliquot of 50 μl was transferred into the tube with support,vortexed for 30 s, centrifugated at 14,500 rpm for 30 s and shaken for30 min at ambient temperature. Supernatant was discarded, the supportwas rinsed with 2×200 μl MeCN, transferred into a synthesiser column,nine dT nucleotides were incorporated and the synthesis was interruptedon the last dT before oxidation. The column was detached fromsynthesiser, drained on a water pump, rinsed with MeCN, and the supportwith attached phosphite was transferred into a plastic tube.

Third aliquot of 50 μl was transferred into the tube with support,vortexed for 30 s, centrifugated at 14,500 rpm for 30 s and shaken for30 min at ambient temperature. Supernatant was discarded, the supportwas rinsed with 2×200 μl MeCN, transferred into a synthesiser column,and the synthesis was resumed for the last dT nucleotide incorporation.

The oligonucleotide was detached from support and protecting groupsremoved by Soln A for 2 h at ambient temperature.

Molecular mass: calc. [M] 6313.49, exp. [M−H] 6310.71.

Example 21. Preparation of a Modified Oligonucleotide5′-d(TTTTTTTTTTTTTTTTTTTpT) (SEQ ID NO: 21) with the PhosphorylGuanidine Group (FIX)

A column containing 5 mg of High Load 5′-DMTr-dT CPG support (110 μmolg⁻¹) was placed into a DNA synthesizer, and automated β-cyanoethylphosphoramidite solid-phase DNA synthesis was started on 0.2 μmol scale.Synthesis was interrupted after four dT incorporations followed by5′-detritylation, phosphoramidite coupling and capping but beforeoxidation. The column was detached from synthesiser, drained on a waterpump, rinsed with MeCN, and the support with attached phosphite wastransferred into a plastic tube.

Soln 1 was prepared by dissolving iodine crystals in dry pyridine to 0.2M concentration (51 mg per ml). Soln 2 was prepared by weighing 9.6 mgof dried guanidine hydrochloride into a plastic tube, adding 85 μl drypyridine, 15 μl DBU, 10 μl BSA, and the tube was vortexed for 5 min andsonicated until clear (ca. 5 min). Aliquots of 20 μl of Soln 1 and 2were mixed in a plastic tube, 10 μl of BSA were added, and after 1 minwait the content was transferred to the tube with polymer. The tube wasvortexed for 30 s, centrifugated at 14,500 rpm for 15 s and shaken for 5min at ambient temperature. Supernatant was discarded, the support wasrinsed with 2×200 μl MeCN, transferred into a synthesiser column, andthe synthesis was resumed until the end of the sequence.

The oligonucleotide was detached from support and protecting groupsremoved by Soln A for 1 h at ambient temperature.

Molecular mass: calc. [M] 6063.05, exp. [M+H] 6065.02, [M−H] 6058.30.

Example 22. Preparation of a Modified Oligodeoxyribonucleotide5′-d(TTTT)tpdT with the N,N,N′,N′-tetramethyl Phosphoryl Guanidine Group(FVIII); t Here and in the Following Example Indicates Position of LNA-TNucleotide

A column containing 5 mg of High Load 5′-DMTr-dT CPG support (110 μmolg⁻¹) was placed into a DNA synthesizer, and automated β-cyanoethylphosphoramidite solid-phase DNA synthesis was started on 0.2 μmol scale.The coupling step for LNA-T incorporation was extended to 6 min.Synthesis was interrupted after 5′-detritylation, LNA-T phosphoramiditecoupling and capping but before oxidation. The column was detached fromsynthesiser, drained on a water pump, rinsed with MeCN, and the supportwith attached phosphite was transferred into a plastic tube.

An aliquot of 40 μl 0.5 M bis(dimethylamino)-1-azidocarbeniumdichlorophosphate was transferred into a plastic tube, 10 μl BSA wasadded and the tube was vortexed for 1 min, centrifugated at 14,500 rpmfor 1 min and left for 30 min at ambient temperature. Then the contentwas transferred into the tube with support, vortexed for 30 s,centrifugated at 14,500 rpm for 30 s and shaken for 1 h at 40° C.Supernatant was discarded, the support was rinsed with 2×200 μl MeCN,transferred into a synthesiser column, and the synthesis was resumeduntil the end of the sequence.

The oligonucleotide was detached from support and protecting groupsremoved by Soln A for 1 h at ambient temperature. An RP-HPLC trace ofthe oligonucleotide is shown in FIG. 3.

Molecular mass: calc. [M] 1888.40, exp. [M+H] 1888.15, [M−H] 1886.86.

Example 23. Preparation of a Modified Oligodeoxyribonucleotide5′-tpd(TTTTT) with the N,N,N′,N′-tetramethyl Phosphoryl Guanidine Group(FVIII); p Indicates Position of Modified Phosphate Group; t IndicatesPosition of LNA-T Nucleotide

A column containing 5 mg of High Load 5′-DMTr-dT CPG support (110 μmolg⁻¹) was placed into a DNA synthesizer, and automated β-cyanoethylphosphoramidite solid-phase DNA synthesis was started on 0.2 μmol scale.The coupling step for LNA-T incorporation was extended to 6 min.Synthesis was interrupted after four cycles of dT incorporation,5′-detritylation, LNA-T phosphoramidite coupling and capping but beforeoxidation. The column was detached from synthesiser, drained on a waterpump, rinsed with MeCN, and the support with attached phosphite wastransferred into a plastic tube.

An aliquot of 40 μl 0.5 M bis(dimethylamino)-1-azidocarbeniumdichlorophosphate was transferred into a plastic tube, 10 μl BSA wasadded and the tube was vortexed for 1 min, centrifugated at 14,500 rpmfor 1 min and left for 30 min at ambient temperature. The content wastransferred into the tube with support, vortexed for 30 s, centrifugatedat 14,500 rpm for 30 s and shaken for 1 h at 40° C. Supernatant wasdiscarded, the support was rinsed with 2×200 μl MeCN, transferred into asynthesiser column, and the synthesis was completed by 5′-detritylation.

The oligonucleotide was detached from support and protecting groupsremoved by Soln A for 1 h at ambient temperature. An RP-HPLC trace ofthe oligonucleotide is shown in FIG. 3.

Molecular mass: calc. [M] 1888.40, exp. [M+H] 1888.27, [M−H] 1887.44.

Example 24. Preparation of a Modified Oligonucleotide5′-d(AACGTCAGGGTCTTCCp)BHQ (SEQ ID NO: 4) with the N,N,N′,N′-tetramethylPhosphoryl Guanidine Group (Formula XIV). Here and in the FollowingExample, BHQ is BlackHole Quencher™ (FXII)

A column containing 5 mg of BHQ CPG support (42 μmol g⁻¹) was placedinto a DNA synthesizer, and automated β-cyanoethyl phosphoramiditesolid-phase DNA synthesis was started on 0.2 μmol scale. Synthesis wasinterrupted after 5′-detritylation, dC phosphoramidite coupling andcapping but before oxidation. The column was detached from synthesiser,drained on a water pump, rinsed with MeCN, and the support with attachedphosphite was transferred into a plastic tube.

An aliquot of 40 μl 0.5 M bis(dimethylamino)-1-azidocarbeniumdichlorophosphate was transferred into a plastic tube, 10 μl BSA wasadded and the tube was vortexed for 1 min, centrifugated at 14,500 rpmfor 1 min and left for 30 min at ambient temperature. Then the contentwas transferred into the tube with support, vortexed for 30 s,centrifugated at 14,500 rpm for 30 s and shaken for 30 min at ambienttemperature. Supernatant was discarded, the support was rinsed with2×200 μl MeCN, transferred into a synthesiser column, and the synthesiswas resumed until the end of the sequence.

The oligonucleotide was detached from support and protecting groupsremoved by Soln A for 12 h at 55° C.

Molecular mass: calc. [M] 5510.92, exp. [M−H] 5509.50.

Example 25. Preparation of a Modified Oligonucleotide 5′-Flu d(GGAAG DDCCCTGACGTTp) BHQ (SEQ ID NO: 5) with the N,N,N′,N′-tetramethylPhosphoryl Guanidine Group (FVIII). DD is 1,12-Dodecanediol Phosphate(FXIII), Flu is 5(6)-Carboxyfluorescein Label (FXIV)

A column containing 5 mg of BHQ CPG support (42 μmol g⁻¹) was placedinto a DNA synthesizer, and automated β-cyanoethyl phosphoramiditesolid-phase DNA synthesis was started on 0.2 μmol scale. Synthesis wasinterrupted after 5′-detritylation, dT phosphoramidite coupling andcapping but before oxidation. The column was detached from synthesiser,drained on a water pump, rinsed with MeCN, and the support with attachedphosphite was transferred into a plastic tube.

An aliquot of 40 μl 0.5 M bis(dimethylamino)-1-azidocarbeniumdichlorophosphate was transferred into a plastic tube, 10 μl BSA wasadded and the tube was vortexed for 1 min, centrifugated at 14,500 rpmfor 1 min and left for 30 min at ambient temperature. Then the contentwas transferred into the tube with support, vortexed for 30 s,centrifugated at 14,500 rpm for 30 s and shaken for 30 min at ambienttemperature. Supernatant was discarded, the support was rinsed with2×200 μl MeCN, transferred into a synthesiser column, and the synthesiswas resumed until the end of the sequence using the corresponding1,12-dodecanediol and fluorescein phosphoramidites as modifiers.

The oligonucleotide was detached from support and protecting groupsremoved by Soln A for 12 h at 55° C.

Molecular mass: calc. [M] 6078.50, exp. [M+H] 6084.38.

Example 26. Preparation of a Modified Oligonucleotide5′-d(TCTCTCpFCCTTCpC) (SEQ ID NO: 6) with the N,N,N′,N′-tetramethylPhosphoryl Guanidine Group (Formula XIV). F Here and in the Next Exampleis 2-hydroxymethyl-3-hydroxytetrahydrofurane (Apurinic/ApyrimidinicSite) Phosphate (FXV)

A column containing 5 mg of 5′-DMTr-dC(Bz) CPG support (110 μmol g⁻¹)was placed into a DNA synthesiser, and automated β-cyanoethylphosphoramidite solid-phase DNA synthesis was started on 0.2 μmol scale.Synthesis was interrupted after 5′-detritylation, dC phosphoramiditecoupling and capping but before oxidation. The column was detached fromsynthesiser, drained on a water pump, rinsed with MeCN, and the supportwith attached phosphite was transferred into a plastic tube.

An aliquot of 80 μl 0.5 M bis(dimethylamino)-1-azidocarbeniumdichlorophosphate was transferred into a plastic tube, 20 μl BSA wasadded and the tube was vortexed for 1 min, centrifugated at 14,500 rpmfor 1 min and left for 30 min at ambient temperature. An aliquot of 50μl was transferred into the tube with support, vortexed for 30 s,centrifugated at 14,500 rpm for 30 s and shaken for 30 min at ambienttemperature. Supernatant was discarded, the support was rinsed with2×200 μl MeCN, transferred into a synthesiser column, five nucleotidesere incorporated including the dF unit until the next dC nucleotide,where the synthesis was interrupted before the oxidation step. Thecolumn was detached from synthesiser, drained on a water pump, rinsedwith MeCN, and the support with attached phosphite was transferred intoa plastic tube.

Second aliquot of 50 μl was transferred into the tube with support,vortexed for 30 s, centrifugated at 14,500 rpm for 30 s and shaken for30 min at ambient temperature. Supernatant was discarded, the supportwas rinsed with 2×200 μl MeCN, transferred into a synthesiser column,and the synthesis was resumed until the end of the sequence.

The oligonucleotide was detached from support and protecting groupsremoved by Soln A for 16 h at ambient temperature.

Molecular mass: calc. [M] 3857.76, exp. [M−H] 3856.74.

Example 27. Preparation of a Modified Oligonucleotide 5′-d(GCGCCAAACpA)(SEQ ID NO: 22) with the 1,3-dimethyl-2-(phosphorylimino)imidazolidineGroup (FXVI)

A column containing 5 mg of 5′-DMTr-dA(Bz) CPG support (110 μmol g⁻¹)was placed into a DNA synthesiser, and automated β-cyanoethylphosphoramidite solid-phase DNA synthesis was started on 0.2 μmol scale.Synthesis was interrupted after 5′-detritylation, dC phosphoramiditecoupling and capping but before oxidation. The column was detached fromsynthesiser, drained on a water pump, rinsed with MeCN, and the supportwith attached phosphite was transferred into a plastic tube.

To 100 μl 1 M 2-azido-4,5-dihydro-1,3-dimethyl-1H-imidazoliumhexafluorophosphate 25 μl BSA and 5 μl triethylamine were added, and thetube was vortexed for 1 min, centrifugated at 14,500 rpm for 1 min andleft for 30 min at ambient temperature. The content was transferred intothe tube with support, vortexed for 30 s, centrifugated at 14,500 rpmfor 30 s and shaken for 1 h at ambient temperature. Supernatant wasdiscarded, the support was rinsed with 2×200 μl MeCN, transferred into asynthesiser column, and the synthesis was resumed until the end of thesequence.

The oligonucleotide was detached from support and protecting groupsremoved by Soln A for 12 h at 55° C.

Molecular mass: calc. [M] 3101.20, exp. [M−H] 3101.55.

Example 28. Preparation of a Modified Oligonucleotide 5′-d(GCGCCAAACpA)(SEQ ID NO: 23) with the N,N′-bis(tetramethylene)-N″-phosphorylGuanidine Group (FXVII)

A column containing 5 mg of 5′-DMTr-dA(Bz) CPG support (110 μmol g⁻¹)was placed into a DNA synthesiser, and automated β-cyanoethylphosphoramidite solid-phase DNA synthesis was started on 0.2 μmol scale.Synthesis was interrupted after 5′-detritylation, dC phosphoramiditecoupling and capping but before oxidation. The column was detached fromsynthesiser, drained on a water pump, rinsed with MeCN, and the supportwith attached phosphite was transferred into a plastic tube.

To 100 μl 1 M azidodipyrrolidinocarbenium hexafluorophosphate 25 μl BSAand 5 μl triethylamine were added, and the tube was vortexed for 1 min,centrifugated at 14,500 rpm for 1 min and left for 30 min at ambienttemperature. The content was transferred into the tube with support,vortexed for 30 s, centrifugated at 14,500 rpm for 30 s and shaken for 1h at ambient temperature. Supernatant was discarded, the support wasrinsed with 2×200 μl MeCN, transferred into a synthesiser column, andthe synthesis was resumed until the end of the sequence.

The oligonucleotide was detached from support and protecting groupsremoved by Soln A for 12 h at 55° C.

Molecular mass: calc. [M] 3155.29, exp. [M−H] 3153.75.

Example 29. Preparation of a Modified oligo-2′-O-Methylribonucleotide5′-AUCGpU with the N,N,N′,N′-tetramethyl Phosphoryl Guanidine Group(FVIII)

A column containing 5 mg of 5′-DMTr-2′-OMe-rU CPG support (40 μmol g⁻¹)was placed into a DNA synthesiser, and automated β-cyanoethylphosphoramidite solid-phase oligo-2′-O-methylribonucleotide synthesiswas started on 0.2 μmol scale. Synthesis was interrupted after5′-detritylation, 2′-OMe-rG phosphoramidite coupling and capping butbefore oxidation. The column was detached from synthesiser, drained on awater pump, rinsed with MeCN, and the support with attached phosphitewas transferred into a plastic tube.

An aliquot of 40 μl 0.5 M bis(dimethylamino)-1-azidocarbeniumdichlorophosphate was transferred into a plastic tube, 10 μl BSA wasadded and the tube was vortexed for 1 min, centrifugated at 14,500 rpmfor 1 min and left for 30 min at ambient temperature. The content wastransferred into the tube with support, vortexed for 30 s, centrifugatedat 14,500 rpm for 30 s and shaken for 30 min at ambient temperature.Supernatant was discarded, the support was rinsed with 2×200 μl MeCN,transferred into a synthesiser column, and the synthesis was resumeduntil the end of the sequence.

The oligonucleotide was detached from support and protecting groupsremoved by Soln A for 16 h at 55° C.

Molecular mass: calc. [M] 1697.28, exp. [M+H] 1697.59, [M−H] 1694.77.

Example 30. Preparation of a Modified oligo-2′-O-methylribonucleotide5′-GCGCCAAACpA (SEQ ID NO: 11) with the N,N,N′,N′-tetramethyl PhosphorylGuanidine Group (FVIII)

A column containing 5 mg of 5′-DMTr-2′-OMe-rA(Bz) CPG support (60 μmolg⁻¹) was placed into a DNA synthesiser, and automated β-cyanoethylphosphoramidite solid-phase oligo-2′-O-methylribonucleotide synthesiswas started on 0.2 μmol scale. Synthesis was interrupted after5′-detritylation, 2′-OMe-rC phosphoramidite coupling and capping butbefore oxidation. The column was detached from synthesiser, drained on awater pump, rinsed with MeCN, and the support with attached phosphitewas transferred into a plastic tube.

An aliquot of 40 μl 0.5 M bis(dimethylamino)-1-azidocarbeniumdichlorophosphate was transferred into a plastic tube, 10 μl BSA wasadded and the tube was vortexed for 1 min, centrifugated at 14,500 rpmfor 1 min and left for 30 min at ambient temperature. The content wastransferred into the tube with support, vortexed for 30 s, centrifugatedat 14,500 rpm for 30 s and shaken for 1 h at 40° C. Supernatant wasdiscarded, the support was rinsed with 2×200 μl MeCN, transferred into asynthesiser column, and the synthesis was resumed until the end of thesequence.

The oligonucleotide was detached from support and protecting groupsremoved by Soln A for 12 h at 55° C.

Molecular mass: calc. [M] 3403.48, exp. [M−H] 3402.55.

Example 31. Preparation of a Modified oligo-2′-O-methylribonucleotide5′-GCGCCAAApCpA (SEQ ID NO: 24) with the N,N,N′,N′-tetramethylPhosphoryl Guanidine Group (FVIII)

A column containing 5 mg of 5′-DMTr-2′-OMe-rA(Bz) CPG support (60 μmolg⁻¹) was placed into a DNA synthesiser, and automated β-cyanoethylphosphoramidite solid-phase oligo-2′-O-methylribonucleotide synthesiswas started on 0.2 μmol scale. Synthesis was interrupted after5′-detritylation, 2′-OMe-rC phosphoramidite coupling and capping butbefore oxidation. The column was detached from synthesiser, drained on awater pump, rinsed with MeCN, and the support with attached phosphitewas transferred into a plastic tube.

An aliquot of 80 μl 0.5 M bis(dimethylamino)-1-azidocarbeniumdichlorophosphate was transferred into a plastic tube, 20 μl BSA wasadded and the tube was vortexed for 1 min, centrifugated at 14,500 rpmfor 1 min and left for 30 min at ambient temperature. An aliquot of 50μl was transferred into the tube with support, vortexed for 30 s,centrifugated at 14,500 rpm for 30 s and shaken for 1 h at 40° C.Supernatant was discarded, the support was rinsed with 2×200 μl MeCN,transferred into a synthesiser column, a 2′-OMe-rU phosphoramidite wascoupled and the synthesis was interrupted before oxidation. The columnwas detached from synthesiser, drained on a water pump, rinsed withMeCN, and the support with attached phosphite was transferred into aplastic tube.

Second aliquot of 50 μl was transferred into the tube with support,vortexed for 30 s, centrifugated at 14,500 rpm for 30 s and shaken for 1h at 40° C. Supernatant was discarded, the support was rinsed with 2×200μl MeCN, transferred into a synthesiser column, and the synthesis wasresumed until the end of the sequence.

The oligonucleotide was detached from support and protecting groupsremoved by Soln A for 12 h at 55° C.

Molecular mass: calc. [M] 3500.64, exp. [M+H] 3199.90, [M−H] 3499.75.

Example 32. Preparation of a modified oligo-2′-O-methylribonucleotidePhosphorothioate5′-G_(s)A_(s)C_(s)A_(s)U_(s)C_(s)C_(s)A_(s)U_(s)U_(s)C_(s)A_(s)A_(s)A_(s)U_(s)G_(s)G_(s)U_(s)UpUpG(SEQ ID NO: 8) with N,N,N′,N′-tetramethyl Phosphoryl Guanidine Group(Formula XIV); _(s) Here and in the Following Examples IndicatesPhosphorothioate Residue (FXVIII)

A column containing 5 mg of 5′-DMTr-2′-OMe-rG(Tac) CPG support (60 μmolg⁻¹) was placed into a DNA synthesiser, and automated β-cyanoethylphosphoramidite solid-phase oligo-2′-O-methylribonucleotide synthesiswas started on 0.2 μmol scale. Synthesis was interrupted after5′-detritylation, 2′-OMe-rU phosphoramidite coupling and capping butbefore oxidation. The column was detached from synthesiser, drained on awater pump, rinsed with MeCN, and the support with attached phosphitewas transferred into a plastic tube.

An aliquot of 80 μl 0.5 M bis(dimethylamino)-1-azidocarbeniumdichlorophosphate was transferred into a plastic tube, 20 μl BSA wasadded and the tube was vortexed for 1 min, centrifugated at 14,500 rpmfor 1 min and left for 30 min at ambient temperature. An aliquot of 50μl was transferred into the tube with support, vortexed for 30 s,centrifugated at 14,500 rpm for 30 s and shaken for 1 h at 40° C.Supernatant was discarded, the support was rinsed with 2×200 μl MeCN,transferred into a synthesiser column, a 2′-OMe-rA phosphoramidite wascoupled and the synthesis was interrupted before oxidation. The columnwas detached from synthesiser, drained on a water pump, rinsed withMeCN, and the support with attached phosphite was transferred into aplastic tube.

Second aliquot of 50 μl was transferred into the tube with support,vortexed for 30 s, centrifugated at 14,500 rpm for 30 s and shaken for 1h at 40° C. Supernatant was discarded, the support was rinsed with 2×200μl MeCN, transferred into a synthesiser column, and the synthesis wasresumed substituting sulfurisation with 0.1 M3-[(dimethylaminomethylene)imino]-3H-1,2,4-dithiazole-3-thione in drypyridine for iodine oxidation until the end of the sequence.

The oligonucleotide was detached from support and protecting groupsremoved by Soln A for 3 h at 70° C.

Molecular mass: calc. [M] 7436.14, exp. [M−H] 7433.89.

Example 33. Preparation of a modified oligo-2′-O-methylribonucleotidePhosphorothioate 5′-FluG_(s)A_(s)C_(s)A_(s)U_(s)C_(s)C_(s)A_(s)U_(s)U_(s)C_(s)A_(s)A_(s)A_(s)U_(s)G_(s)G_(s)U_(s)UpUpG(SEQ ID NO: 8) with N,N,N′,N′-tetramethyl Phosphoryl Guanidine Group(FVIII); Flu Indicates 5′-Fluorescein Label (FXIXV); _(s) IndicatesPhosphorothioate Residue (FXVIII)

A column containing 5 mg of 5′-DMTr-2′-OMe-rG(Tac) CPG support (60 μmolg⁻¹) was placed into a DNA synthesiser, and automated β-cyanoethylphosphoramidite solid-phase oligo-2′-O-methylribonucleotide synthesiswas started on 0.2 μmol scale. Synthesis was interrupted after5′-detritylation, 2′-OMe-rU phosphoramidite coupling and capping butbefore oxidation. The column was detached from synthesiser, drained on awater pump, rinsed with MeCN, and the support with attached phosphitewas transferred into a plastic tube.

An aliquot of 80 μl 0.5 M bis(dimethylamino)-1-azidocarbeniumdichlorophosphate was transferred into a plastic tube, 20 μl BSA wasadded and the tube was vortexed for 1 min, centrifugated at 14,500 rpmfor 1 min and left for 30 min at ambient temperature. An aliquot of 50μl was transferred into the tube with support, vortexed for 30 s,centrifugated at 14,500 rpm for 30 s and shaken for 1 h at 40° C.Supernatant was discarded, the support was rinsed with 2×200 μl MeCN,transferred into a synthesiser column, a 2′-OMe-rA phosphoramidite wascoupled and the synthesis was interrupted before oxidation. The columnwas detached from synthesiser, drained on a water pump, rinsed withMeCN, and the support with attached phosphite was transferred into aplastic tube.

Second aliquot of 50 μl was transferred into the tube with support,vortexed for 30 s, centrifugated at 14,500 rpm for 30 s and shaken for 1h at 40° C. Supernatant was discarded, the support was rinsed with 2×200μl MeCN, transferred into a synthesiser column, and the synthesis wasresumed substituting sulfurisation with 0.1 M3-[(dimethylaminomethylene)imino]-3H-1,2,4-dithiazole-3-thione in drypyridine for iodine oxidation until the end of the sequence. Thesynthesis was completed by the incorporation of the correspondingfluorescein phosphoramidite in DMTr ON mode.

The oligonucleotide was detached from support and protecting groupsremoved by Soln A for 3 h at 70° C.

Molecular mass: calc. [M] 8003.63, exp. [M−H] 8001.25.

Example 34. Preparation of a Modified oligo-2′-O-methylribonucleotidePhosphorothioate 5′-FluG_(s)G_(s)C_(s)C_(s)A_(s)A_(s)A_(s)C_(s)C_(s)U_(s)C_(s)C_(s)G_(s)C_(s)UpUpACpCpU(SEQ ID NO: 9) with N,N,N′,N′-tetramethyl Phosphoryl Guanidine Group(FVIII); Flu Indicates 5′-Fluorescein Label (FXIX); _(s) IndicatesPhosphorothioate Residue (FXVIII)

A column containing 5 mg of 5′-DMTr-2′-OMe-rU CPG support (40 μmol g⁻¹)was placed into a DNA synthesiser, and automated β-cyanoethylphosphoramidite solid-phase oligo-2′-O-methylribonucleotide synthesiswas started on 0.2 μmol scale. Synthesis was interrupted after5′-detritylation, 2′-OMe-rC phosphoramidite coupling and capping butbefore oxidation. The column was detached from synthesiser, drained on awater pump, rinsed with MeCN, and the support with attached phosphitewas transferred into a plastic tube.

An aliquot of 160 μl 0.5 M bis(dimethylamino)-1-azidocarbeniumdichlorophosphate was transferred into a plastic tube, 40 μl BSA wasadded and the tube was vortexed for 1 min, centrifugated at 14,500 rpmfor 1 min and left for 30 min at ambient temperature. An aliquot of 50μl was transferred into the tube with support, vortexed for 30 s,centrifugated at 14,500 rpm for 30 s and shaken for 1 h at 40° C.Supernatant was discarded, the support was rinsed with 2×200 μl MeCN,transferred into a synthesiser column, a 2′-OMe-rC phosphoramidite wascoupled and the synthesis was interrupted before oxidation. The columnwas detached from synthesiser, drained on a water pump, rinsed withMeCN, and the support with attached phosphite was transferred into aplastic tube.

Second aliquot of 50 μl was transferred into the tube with support,vortexed for 30 s, centrifugated at 14,500 rpm for 30 s and shaken for 1h at 40° C. Supernatant was discarded, the support was rinsed with 2×200μl MeCN, transferred into a synthesiser column, a 2′-OMe-rAphosphoramidite was coupled followed by 2′-OMe-rU and the synthesis wasinterrupted before the last oxidation step. The column was detached fromsynthesiser, drained on a water pump, rinsed with MeCN, and the supportwith attached phosphite was transferred into a plastic tube.

Third aliquot of 50 μl was transferred into the tube with support,vortexed for 30 s, centrifugated at 14,500 rpm for 30 s and shaken for1.5 h at 45° C. Supernatant was discarded, the support was rinsed with2×200 μl MeCN, transferred into a synthesiser column, a 2′-OMe-rUphosphoramidite was coupled and the synthesis was interrupted beforeoxidation. The column was detached from synthesiser, drained on a waterpump, rinsed with MeCN, and the support with attached phosphite wastransferred into a plastic tube.

Fourth aliquot of 50 μl was transferred into the tube with support,vortexed for 30 s, centrifugated at 14,500 rpm for 30 s and shaken for1.5 h at 45° C. Supernatant was discarded, the support was rinsed with2×200 μl MeCN, transferred into a synthesiser column, and the synthesiswas resumed substituting sulfurisation with 0.1 M3-[(dimethylaminomethylene)imino]-3H-1,2,4-dithiazole-3-thione in drypyridine for iodine oxidation until the end of the sequence. Thesynthesis was completed by the incorporation of the correspondingfluorescein phosphoramidite in DMTr ON mode.

The oligonucleotide was detached from support and protecting groupsremoved by Soln A for 3 h at 70° C.

Molecular mass: calc. [M] 7763.48, exp. [M−H] 7759.37.

Example 35. Preparation of a mixed LNA-oligo-2′-O-methylribonucleotidePhosphorothioate 5′-FluG_(s)g_(s)C_(s)g_(s)A_(s)a_(s)A_(s)C_(s)c_(s)U_(s)C_(s)C_(s)c_(s)C_(s)UpUpaCpCpU(SEQ ID NO: 10) with N,N,N′,N′-tetramethyl Phosphoryl Guanidine Group(FVIII); Flu Indicates 5′-Fluorescein Label (FXIX); _(s) IndicatesPhosphorothioate Residue (FXVIII). LNA Nucleotides A, 5-Me-C and G areShown by Lowercase Letters a, c and g, Respectively

A column containing 5 mg of 5′-DMTr-2′-OMe-rU CPG support (40 μmol g⁻¹)was placed into a DNA synthesiser, and automated β-cyanoethylphosphoramidite solid-phase oligo-2′-O-methylribonucleotide synthesiswas started on 0.2 μmol scale. Synthesis was interrupted after5′-detritylation, 2′-OMe-rC phosphoramidite coupling and capping butbefore oxidation. The column was detached from synthesiser, drained on awater pump, rinsed with MeCN, and the support with attached phosphitewas transferred into a plastic tube.

An aliquot of 160 μl 0.5 M bis(dimethylamino)-1-azidocarbeniumdichlorophosphate was transferred into a plastic tube, 40 μl BSA wasadded and the tube was vortexed for 1 min, centrifugated at 14,500 rpmfor 1 min and left for 30 min at ambient temperature. An aliquot of 50μl was transferred into the tube with support, vortexed for 30 s,centrifugated at 14,500 rpm for 30 s and shaken for 1 h at 40° C.Supernatant was discarded, the support was rinsed with 2×200 μl MeCN,transferred into a synthesiser column, a 2′-OMe-rC phosphoramidite wascoupled and the synthesis was interrupted before oxidation. The columnwas detached from synthesiser, drained on a water pump, rinsed withMeCN, and the support with attached phosphite was transferred into aplastic tube.

Second aliquot of 50 μl was transferred into the tube with support,vortexed for 30 s, centrifugated at 14,500 rpm for 30 s and shaken for 1h at 40° C. Supernatant was discarded, the support was rinsed with 2×200μl MeCN, transferred into a synthesiser column, an LNA-A phosphoramiditewas coupled followed by 2′-OMe-rU and the synthesis was interruptedbefore the last oxidation step. The column was detached fromsynthesiser, drained on a water pump, rinsed with MeCN, and the supportwith attached phosphite was transferred into a plastic tube.

Third aliquot of 50 μl was transferred into the tube with support,vortexed for 30 s, centrifugated at 14,500 rpm for 30 s and shaken for1.5 h at 45° C. Supernatant was discarded, the support was rinsed with2×200 μl MeCN, transferred into a synthesiser column, a 2′-OMe-rUphosphoramidite was coupled and the synthesis was interrupted beforeoxidation. The column was detached from synthesiser, drained on a waterpump, rinsed with MeCN, and the support with attached phosphite wastransferred into a plastic tube.

Fourth aliquot of 50 μl was transferred into the tube with support,vortexed for 30 s, centrifugated at 14,500 rpm for 30 s and shaken for1.5 h at 45° C. Supernatant was discarded, the support was rinsed with2×200 μl MeCN, transferred into a synthesiser column, and the synthesiswas resumed substituting sulfurisation with 0.1 M3-[(dimethylaminomethylene)imino]-3H-1,2,4-dithiazole-3-thione in drypyridine for iodine oxidation and using LNA and 2′-OMe phosphoramiditesuntil the end of the sequence. The synthesis was completed by theincorporation of the corresponding fluorescein phosphoramidite in DMTrON mode.

The oligonucleotide was detached from support and protecting groupsremoved by Soln A for 3 h at 70° C.

Molecular mass: calc. [M] 7793.47, exp. [M−H] 7792.92.

Example 36. Preparation of a Modified oligo-2′-O-methylribonucleotide5′-UUUUUpU with the 1,3-dimethyl-2-(phosphorylimino)imidazolidine Group(FXVI)

A column containing 5 mg of 5′-DMTr-2′-OMe-rU CPG support (40 μmol g⁻¹)was placed into a DNA synthesiser, and automated β-cyanoethylphosphoramidite solid-phase oligo-2′-O-methylribonucleotide synthesiswas started on 0.2 μmol scale. Synthesis was interrupted after5′-detritylation, 2′-OMe-rU phosphoramidite coupling and capping butbefore oxidation. The column was detached from synthesiser, drained on awater pump, rinsed with MeCN, and the support with attached phosphitewas transferred into a plastic tube.

To 100 μl 1 M 2-azido-4,5-dihydro-1,3-dimethyl-1H-imidazoliumhexafluorophosphate 25 μl BSA and 5 μl triethylamine were added, and thetube was vortexed for 1 min, centrifugated at 14,500 rpm for 1 min andleft for 30 min at ambient temperature. The content was transferred intothe tube with support, vortexed for 30 s, centrifugated at 14,500 rpmfor 30 s and shaken for 1 h at ambient temperature. Supernatant wasdiscarded, the support was rinsed with 2×200 μl MeCN, transferred into asynthesiser column, and the synthesis was resumed until the end of thesequence.

The oligonucleotide was detached from support and protecting groupsremoved by Soln A for 1 h at ambient temperature. An RP-HPLC trace ofthe oligonucleotide is shown in FIG. 4.

Molecular mass: calc. [M] 1954.37, exp. [M−H] 1953.37.

Example 37. Preparation of a Modified oligo-2′-O-methylribonucleotide5′-UUUUUpU with the N,N′-bis(tetramethylene)-N″-phosphoryl GuanidineGroup (FXVII)

A column containing 5 mg of 5′-DMTr-2′-OMe-rU CPG support (40 μmol g⁻¹)was placed into a DNA synthesiser, and automated β-cyanoethylphosphoramidite solid-phase oligo-2′-O-methylribonucleotide synthesiswas started on 0.2 μmol scale. Synthesis was interrupted after5′-detritylation, 2′-OMe-rU phosphoramidite coupling and capping butbefore oxidation. The column was detached from synthesiser, drained on awater pump, rinsed with MeCN, and the support with attached phosphitewas transferred into a plastic tube.

To 100 μl 1 M azidodipyrrolidinocarbenium hexafluorophosphate 25 μl BSAand 5 μl triethylamine were added, and the tube was vortexed for 1 min,centrifugated at 14,500 rpm for 1 min and left for 30 min at ambienttemperature. The content was transferred into the tube with support,vortexed for 30 s, centrifugated at 14,500 rpm for 30 s and shaken for 1h at ambient temperature. Supernatant was discarded, the support wasrinsed with 2×200 μl MeCN, transferred into a synthesiser column, andthe synthesis was resumed until the end of the sequence.

The oligonucleotide was detached from support and protecting groupsremoved by Soln A for 1 h at ambient temperature. An RP-HPLC trace ofthe oligonucleotide is shown in FIG. 4.

Molecular mass: calc. [M] 2008.46, exp. [M−H] 2007.57.

Example 38. Preparation of a Modified Oligonucleotide 5′-d(TTTTTpT) withthe N,N-dimethyl Phosphoryl Guanidine Group (FXX)

A column containing 5 mg of High Load 5′-DMTr-dT CPG support (110 μmolg⁻¹) was placed into a DNA synthesizer, and automated β-cyanoethylphosphoramidite solid-phase DNA synthesis was started on 0.2 μmol scale.Synthesis was interrupted after 5′-detritylation, phosphoramiditecoupling and capping but before oxidation. The column was detached fromsynthesiser, drained on a water pump, rinsed with MeCN, and the supportwith attached phosphite was transferred into a plastic tube.

To 12 mg of (chloromethylene)dimethyliminium chloride and 4 mg (1.1equiv) NaN₃ in a plastic tube, 100 μl dry MeCN were added and the tubewas shaken for 2 h at 30° C., centrifugated at 14,500 rpm for 5 min, 25μl BSA were added, the tube vortexed for 1 min, centrifugated at 14,500rpm for 1 min and the supernatant was transferred into the tube withsupport, vortexed for 30 s, centrifugated at 14,500 rpm for 30 s andshaken for 30 min at ambient temperature. Supernatant was discarded, thesupport was rinsed with 2×200 μl MeCN, transferred into a synthesisercolumn, and β-cyanoethyl phosphoramidite solid-phase synthesis wasresumed until the end of the sequence.

The oligonucleotide was detached from support and protecting groupsremoved by Soln A for 18 h at 55° C.

Molecular mass: calc. [M] 1832.22, exp. [M−H] 1831.57.

Example 39. Preparation of a Modified Oligonucleotide 5′-d(TTTTTpT) withthe N-phosphoryl Formamidine Group (FXXI)

A column containing 5 mg of High Load 5′-DMTr-dT CPG support (110 μmolg⁻¹) was placed into a DNA synthesizer, and automated β-cyanoethylphosphoramidite solid-phase DNA synthesis was started on 0.2 μmol scale.Synthesis was interrupted after 5′-detritylation, phosphoramiditecoupling and capping but before oxidation. The column was detached fromsynthesiser, drained on a water pump, rinsed with MeCN, and the supportwith attached phosphite was transferred into a plastic tube.

Soln 1 was prepared by dissolving iodine crystals in dry pyridine to 0.2M concentration (51 mg per ml). Soln 2 was prepared by weighing 40 mg ofdried formamidine hydrochloride into a plastic tube, adding 375 μl drypyridine, 75 μl DBU and 50 μl BSA, and the tube was vortexed for 3-4 minand sonicated until clear (ca. 3-4 min). Aliquots of 20 μl of Soln 1 and2 were mixed in a plastic tube, 10 μl of BSA were added, and after 1 minwait the content was transferred to the tube with polymer. The tube wasvortexed for 30 s, centrifugated at 14,500 rpm for 15 s and shaken for 5min at ambient temperature. Supernatant was discarded, the support wasrinsed with 2×200 μl MeCN, transferred into a synthesiser column, andβ-cyanoethyl phosphoramidite solid-phase synthesis was resumed until theend of the sequence.

The oligonucleotide was detached from support and protecting groupsremoved by Soln A for 1 h at ambient temperature.

Molecular mass: calc. [M] 1789.27, exp. [M−H] 1788.60.

Example 40. Preparation of a Modified Oligonucleotide 5′-d(TTTTTpT) withthe N-cyanoimino Phosphate Group (FXXII)

A column containing 5 mg of High Load 5′-DMTr-dT CPG support (110 μmolg⁻¹) was placed into a DNA synthesizer, and automated β-cyanoethylphosphoramidite solid-phase DNA synthesis was started on 0.2 umol scale.Synthesis was interrupted after 5′-detritylation, phosphoramiditecoupling and capping but before oxidation. The column was detached fromsynthesiser, drained on a water pump, rinsed with MeCN, and the supportwith attached phosphite was transferred into a plastic tube.

To 20 μl 0.25 M solution of cyanogen azide in dry MeCN prepared asdescribed by McMurry [51] 5 μl BSA were added and the tube was kept for15 min at ambient temperature, the content was transferred into the tubewith support, vortexed for 30 s, centrifugated at 14,500 rpm for 15 sand shaken for 5 min at ambient temperature. Supernatant was discarded,the support was rinsed with 2×200 μl MeCN, transferred into asynthesiser column, and β-cyanoethyl phosphoramidite solid-phasesynthesis was resumed until the end of the sequence.

The oligonucleotide was detached from support and protecting groupsremoved by Soln B for 1 h at 70° C.

Molecular mass: calc. [M] 1787.25, exp. [M+H] 1787.27, [M−H] 1785.19.

Example 41. Preparation of a Modified Oligonucleotide 5′-d(TpTTTTT) withthe N-cyanoimino Phosphate Group (FXXII)

A column containing 5 mg of High Load 5′-DMTr-dT CPG support (110 μmolg⁻¹) was placed into a DNA synthesizer, and automated β-cyanoethylphosphoramidite solid-phase DNA synthesis was started on 0.2 μmol scale.Synthesis was interrupted after four cycles of dT incorporation,5′-detritylation, dT phosphoramidite coupling and capping but beforeoxidation. The column was detached from synthesiser, drained on a waterpump, rinsed with MeCN, and the support with attached phosphite wastransferred into a plastic tube.

To 20 μl 0.25 M solution of cyanogen azide in dry MeCN prepared asdescribed by McMurry [51] 5 μl BSA were added and the tube was kept for15 min at ambient temperature, the content was transferred into the tubewith support, vortexed for 30 s, centrifugated at 14,500 rpm for 15 sand shaken for 5 min at ambient temperature. Supernatant was discarded,the support was rinsed with 2×200 μl MeCN, transferred into asynthesiser column, and β-cyanoethyl phosphoramidite solid-phasesynthesis was resumed until the end of the sequence.

The oligonucleotide was detached from support and protecting groupsremoved by Soln B for 1 h at 70° C. An RP-HPLC of the oligonucleotide isshown in FIG. 5.

Molecular mass: calc. [M] 1787.25, exp. [M+H] 1787.19, [M−H] 1785.11.

Example 42. Preparation of a Modified Nucleoside dTp with the3′-N,N′-bis(tetramethylene)-N″-guanidinophosphate Group (FXXIII)

A column containing 5 mg of 3′-phosphate CPG support (30-40 μmol g⁻¹)was placed into a DNA synthesizer, and automated β-cyanoethylphosphoramidite solid-phase DNA synthesis was started on 0.2 μmol scale.Synthesis was interrupted after 5′-detritylation, dT phosphoramiditecoupling and capping but before oxidation. The column was detached fromsynthesiser, drained on a water pump, rinsed with MeCN, and the supportwith attached phosphite was transferred into a plastic tube.

To 100 μl 1 M azidodipyrrolidinocarbenium hexafluorophosphate 25 μl BSAand 5 μl triethylamine were added, and the tube was vortexed for 1 min,centrifugated at 14,500 rpm for 1 min and left for 30 min at ambienttemperature. The content was transferred into the tube with support,vortexed for 30 s, centrifugated at 14,500 rpm for 30 s and shaken for 1h at ambient temperature. Supernatant was discarded, the support wasrinsed with 2×200 μl MeCN, transferred into a synthesiser column, andthe synthesis was completed by 5′-detritylation.

The nucleotide was detached from support by Soln A for 1 h at ambienttemperature. Molecular mass: calc. [M] 471.45, exp. [M−H] 471.08.

Example 43. Preparation of a Modified Oligonucleotide 5′-d(TTTTTT)p withthe 3′-N,N′-bis(tetramethylene)-N″-guanidinophosphate Group (FXXIII)

A column containing 5 mg of 3′-phosphate CPG support (30-40 μmol g⁻¹)was placed into a DNA synthesizer, and automated β-cyanoethylphosphoramidite solid-phase DNA synthesis was started on 0.2 μmol scale.Synthesis was interrupted after 5′-detritylation, dT phosphoramiditecoupling and capping but before oxidation. The column was detached fromsynthesiser, drained on a water pump, rinsed with MeCN, and the supportwith attached phosphite was transferred into a plastic tube.

To 100 μl 1 M azidodipyrrolidinocarbenium hexafluorophosphate 25 μl BSAand 5 μl triethylamine were added, and the tube was vortexed for 1 min,centrifugated at 14,500 rpm for 1 min and left for 30 min at ambienttemperature. The content was transferred into the tube with support,vortexed for 30 s, centrifugated at 14,500 rpm for 30 s and shaken for 1h at ambient temperature. Supernatant was discarded, the support wasrinsed with 2×200 μl MeCN, transferred into a synthesiser column, andthe synthesis was resumed until the end of the sequence.

The nucleotide was detached from support by Soln A for 1 h at ambienttemperature. An RP-HPLC of the oligonucleotide is shown in FIG. 6.

Molecular mass: calc. [M] 1992.44, exp. [M−H] 1991.46.

Example 44. Preparation of a Modified Thymidine Nucleoside with the5′-N,N′-bis(tetramethylene)-N″-phosphorylguanidine Group and aFluorescein Label (FXXIV)

A column containing 5 mg of High Load 5′-DMTr-dT CPG support (110 μmolg⁻¹) was placed into a DNA synthesizer, and automated β-cyanoethylphosphoramidite solid-phase DNA synthesis was started on 0.2 μmol usingthe same fluorescein phosphoramidite as in Example 25. Synthesis wasinterrupted before oxidation, the column was detached from synthesiser,drained on a water pump, rinsed with MeCN, and the support with attachedphosphite was transferred into a plastic tube.

To 100 μl 1 M azidodipyrrolidinocarbenium hexafluorophosphate 25 μl BSAand 5 μl triethylamine were added, and the tube was vortexed for 1 min,centrifugated at 14,500 rpm for 1 min and left for 30 min at ambienttemperature. The content was transferred into the tube with support,vortexed for 30 s, centrifugated at 14,500 rpm for 30 s and shaken for 1h at ambient temperature. Supernatant was discarded, the support wasrinsed with 2×200 μl MeCN and dried on air for 5 min.

The nucleotide was detached from support by Soln A for 1 h at ambienttemperature. An RP-HPLC of the oligonucleotide is shown in FIG. 6.

Molecular mass: calc. [M] 1263.32, exp. [M−H] 1262.22.

Example 45. Preparation of a Modified Oligonucleotide 5′-Flu pd(TTTTTT)with the 5′-N,N′-bis(tetramethylene)-N″-phosphoryl Guanidine Group and aFluorescein Label (FXXIV)

A column containing 5 mg of High Load 5′-DMTr-dT CPG support (110 μmolg⁻¹) was placed into a DNA synthesizer, and automated β-cyanoethylphosphoramidite solid-phase DNA synthesis was started on 0.2 μmol.Synthesis was interrupted after incorporation of five dT residues and afluorescein label Flu′ (XXV) but before the last oxidation step. Thecolumn was detached from synthesiser, drained on a water pump, rinsedwith MeCN, and the support with attached phosphite was transferred intoa plastic tube.

To 100 μl 1 M azidodipyrrolidinocarbenium hexafluorophosphate 25 μl BSAand 5 μl triethylamine were added, and the tube was vortexed for 1 min,centrifugated at 14,500 rpm for 1 min and left for 30 min at ambienttemperature. The content was transferred into the tube with support,vortexed for 30 s, centrifugated at 14,500 rpm for 30 s and shaken for 1h at ambient temperature. Supernatant was discarded, the support wasrinsed with 2×200 μl MeCN and dried on air for 5 min.

The oligonucleotide was detached from support by Soln A for 1 h atambient temperature. An RP-HPLC of the oligonucleotide is shown in FIG.6.

Molecular mass: calc. [M] 2784.31, exp. [M−H] 2782.96.

Example 46. Preparation of a Modified Hybrid DNA-RNA Oligonucleotide5′-d(TTTT)rUpdT with the 1,3-dimethyl-2-(phosphorylimino)imidazolidineGroup (FXVI)

A column containing 5 mg of High Load 5′-DMTr-dT CPG support (110 μmolg⁻¹) was placed into a DNA synthesiser, and automated β-cyanoethylphosphoramidite solid-phase oligonucleotide synthesis was started on 0.2μmol scale. Synthesis was interrupted after 5′-detritylation, 2′-TOM-rUphosphoramidite coupling and capping but before oxidation. The columnwas detached from synthesiser, drained on a water pump, rinsed withMeCN, and the support with attached phosphite was transferred into aplastic tube.

To 100 μl 1 M 2-azido-4,5-dihydro-1,3-dimethyl-1H-imidazoliumhexafluorophosphate 25 μl BSA and 5 μl triethylamine were added, and thetube was vortexed for 1 min, centrifugated at 14,500 rpm for 1 min andleft for 30 min at ambient temperature. The content was transferred intothe tube with support, vortexed for 30 s, centrifugated at 14,500 rpmfor 30 s and shaken for 1 h at ambient temperature. Supernatant wasdiscarded, the support was rinsed with 2×200 μl MeCN, transferred into asynthesiser column, and the synthesis was resumed until the end of thesequence.

The oligonucleotide was detached from support and protecting groupsremoved by Soln A for 1 h at ambient temperature. The TOM group was thenremoved by triethylamine trihydrofluoride-triethylamine-NMP (4:3:6v/v/v) at 65° C. for 2 h.

Molecular mass: calc. [M] 1860.34, exp. [M−H] 1859.21.

Example 47. A Protocol for Preparation of oligodeoxyribonucleotides5′-d(TTTTTpT), 5′-d(TTTTpTpT), 5′-d(TTTpTpTpT), 5′-d(TTpTpTpTpT),5′-d(TpTpTpTpTpT) vi 5′-d(GpCpGpCpCpApApApCpA) (SEQ ID NO: 1) modifiedwith 1,3-dimethyl-N-phosphorylimino-2-imidazolidine Groups (FXVI) Usingan Automated DNA Synthesiser

A column containing 5 mg of either 5′-DMTr-dT or dA CPG support (35-110μmol g⁻¹) was placed into a DNA synthesizer, and automated β-cyanoethylphosphoramidite solid-phase DNA synthesis was started on 0.2 μmol scalesubstituting treatment with 1 M2-azido-4,5-dihydro-1,3-dimethyl-1H-imidazolium hexafluorophosphate indry acetonitrile for iodine oxidation for the corresponding positions(p) within the sequence.

Oligonucleotides were detached from support by Soln A for 12 h at 55° C.for oligonucleotide 5′-d(GpCpGpCpCpApApApCpA) (SEQ ID NO: 1) or for 1 hat ambient temperature for oligothymidylates. An RP-HPLC of theoligonucleotides is shown in FIGS. 7 and 8.

Molecular Masses:

5′-d(TTTTTpT)—calc. [M] 1858.37, exp. [M−H] 1857.57;

5′-d(TTTTpTpT)—calc. [M] 1953.32, exp. [M−H] 1952.57;

5′-d(TTTpTpTpT)—calc. [M] 2048.67, exp. [M−H] 2047.77;

5′-d(TTpTpTpTpT)—calc. [M] 2143.82, exp. [M−H] 2142.77;

5′-d(TpTpTpTpTpT)—calc. [M] 2238.96, exp. [M−H] 2237.97;

5′-d(GpCpGpCpCpApApApCpA) (SEQ ID NO: 1)—calc. [M] 3862.38, exp. [M−H]3860.50.

REFERENCES

A number of publications are cited herein in order more fully todescribe and disclose the invention and the state of the art to whichthe invention pertains. Full citations for these references are providedbelow. Each of these references is incorporated herein by reference inits entirety into the present disclosure, to the same extent as if eachindividual reference was specifically and individually indicated to beincorporated by reference.

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The invention claimed is:
 1. A compound of Formula (I)

wherein Z is selected from —O⁻, —S⁻, —Se⁻, —(N⁻)R^(N), or a protectinggroup (PG); X is selected from the 5′-O— of a nucleoside oroligonucleotide and Y is selected from the 3′-O— of a nucleosideoligonucleotide; —H, —OH, —SH, —NHR^(N), —O-PG, or —S-PG, wherein PG isa protecting group; a linker, a monophosphate or diphosphate, or a labelor quencher; or Y is selected from the 3′-O— of a nucleoside oroligonucleotide and X is selected from the 5′-O— of a nucleoside,oligonucleotide, —H, —OH, —SH, —NHR^(N), —O-PG, or —S-PG, wherein PG isa protecting group; a linker, a monophosphate or diphosphate, or a labelor quencher; R¹ is selected from —NR^(1A)R^(1B), —OR³, —SR³, —H, —S(O)H,—S(O)R³, —S(O)₂H, —S(O)₂R³, —S(O)₂NH₂, —S(O)₂NHR³, —S(O)₂NR³ ₂,—C₁₋₁₀alkyl, —C₂₋₁₀alkenyl, —C₂₋₁₀alkynyl, or —C₆₋₁₀ aryl; R² isselected from —H, —NR^(2A)R^(2B), —OR³, —SR³, —CN, —S(O)H, —S(O)R³,—S(O)₂H, —S(O)₂R³, —S(O)₂NH₂, —S(O)₂NHR³, —S(O)₂NR³ ₂, —C₁₋₁₀alkyl,—C₂₋₁₀alkenyl, —C₂₋₁₀alkynyl, or —C₆₋₁₀aryl; wherein each R^(1A),R^(1B), R^(2A), and R^(2B) is independently selected from —H,—C₁₋₁₀alkyl, —C₂₋₁₀alkenyl, —C₂₋₁₀alkynyl, or —C₆₋₁₀aryl; optionally,wherein R^(1A) and R^(2A) together form an alkylene chain of 2-4 atomsin length; optionally, wherein R^(1A) and R^(1B), together with the atomto which they are bound, form a 5-8 membered heterocyclic substituentmoiety, selected from the group consisting of N-pyrrolidinyl,N-piperidinyl, N-azepanyl, or N-azocanyl; optionally, wherein R^(2A) andR^(2B), together with the atom to which they are bound, form a 5-8membered heterocyclic substituent moiety, selected from the groupconsisting of N-pyrrolidinyl, N-piperidinyl, N-azepanyl, or N-azocanyl;R³ is selected from —C₁₋₁₀alkyl, —C₂₋₁₀alkenyl, —C₂₋₁₀alkynyl, or—C₆₋₁₀aryl; wherein the linker is selected from the group consisting ofsuccinyl, diglycolyl, oxalyl, hydroquinone-O,O′-diacetyl (Q-linker),phthaloyl, 4,5-dichlorophthaloyl, malonyl, glutaryl, diisopropylsilyland 1,1,3,3-tetraisopropyldisiloxane-1,3-diyl, ordodecane-12-oxy-1-phosphatidyl, the label is fluorescein-derivedsubstituent, the quencher is2-((2-hydroxy-ethyl)-{4-[2-methoxy-5-methyl-4-(4-methyl-2-nitro-phenylazo)-phenylazo]-phenyl}-amino)-ethanol-derivedsubstituent; wherein R^(N) is —H or —C₁₋₄alkyl; and when Z is PG, thecompound is a salt with anion counterpart selected from the groupconsisting of I⁻, Br⁻, Cl⁻, N-succinimido ((CH₂)₂(CO)₂N⁻), CCl₃ ⁻, CBr₃⁻, CI₃ ⁻, CHI₂ ⁻, trifluoromethanesulfonate (CF₃SO₃ ⁻),p-toluenesulphonate (C₇H₇SO₃ ⁻), dichlorophosphate (PO₂Cl₂ ⁻),perchlorate (ClO₄ ⁻), tetrafluoroborate (BF₄ ⁻), tetraphenylborate (BPh₄⁻), or hexafluorophosphate (PF₆ ⁻).
 2. The compound of claim 1, whereinR¹ is —NR^(1A)R^(1B), —OR³, or —SR³.
 3. The compound of claim 1, whereinR¹ is NR^(1A)R^(1B).
 4. The compound of claim 1, wherein R² is —H,—NR^(2A)R^(2B) or —OR³.
 5. The compound of claim 1, wherein R² is—NR^(2A)R^(2B).
 6. The compound of claim 1, wherein each R^(1A), R^(1B),R^(2A), and R^(2B) is independently selected from —H or —C₁₋₁₀alkyl. 7.The compound of claim 6, wherein each R^(1A), R^(1B), R^(2A), and R^(2B)are each methyl.
 8. The compound of claim 1, wherein R^(1A) and R^(2A)together from form an alkylene chain of 2-4 atoms in length and R^(1B)and R^(2B) are each independently selected from —H or —C₁₋₄alkyl.
 9. Thecompound of claim 8, wherein R^(1A) and R^(2A) together from —CH₂—CH₂—and R^(1B) and R^(2B) are each independently selected from —H andmethyl.
 10. The compound of claim 1, wherein R^(1A) and R^(1B), togetherwith the atom to which they are bound, form a 5-8 membered heterocyclicsubstituent moiety, selected from the group consisting N pyrrolidinyl,N-piperidinyl, N-azepanyl, or N-azocanyl.
 11. The compound of claim 10,wherein R^(2A) and R^(2B), together with the atom to which they arebound, form a 5-8 membered heterocyclic substituent moiety, selectedfrom the group consisting of N-pyrrolidinyl, N-piperidinyl, N-azepanyl,or N-azocanyl.
 12. The compound of claim 1, wherein the heterocyclicsubstituent moiety is a N-pyrrolidinyl.
 13. A method of synthesizing thecompound according to claim 1, the method comprising reaction of aphosphorous acid derivative of formula

wherein Z is a protecting group (PG); X is selected from the 5′-O— of anucleoside oligonucleotide and Y is selected from the 3′-O— of anucleoside, oligonucleotide; —H, —OH, —SH, —NHR^(N), —O-PG, —S-PG,wherein PG is a protecting group; a linker, a monophosphate ordiphosphate, or a label or quencher; or Y is selected from the 3′-O— ofa nucleoside, oligonucleotide and X is selected from the 5′-O— of anucleoside, oligonucleotide, —H, —OH, —SH, —NHR^(N), —O-PG, or —S-PG,wherein PG is a protecting group; a linker, a monophosphate ordiphosphate, or a label or quencher, with an imino derivative HN═CR¹R²or an N-silylated derivative R^(Si) ₃SiN═CR¹R² in the presence of anoxidant, wherein R¹ is selected from —NR^(1A)R^(1B), —0R³, —SR³, —H,—S(O)H, —S(O)R³, —S(O)₂H, —S(O)₂R³, —S(O)₂NH₂, —S(O)₂NHR³, —S(O)₂NR³ ₂,—C₁₋₁₀alkyl, —C₂₋₁₀alkenyl, —C₂₋₁₀alkynyl, or —C₆₋₁₀ aryl; R² isselected from —H, —NR^(2A)R^(2B), —OR³, —SR³, CN, —S(O)H, —S(O)R³,—S(O)₂H, —S(O)₂R³, —S(O)₂NH₂, —S(O)₂NHR³, —S(O)₂NR³ ₂, —C₁₋₁₀alkyl,—C₂₋₁₀alkenyl, —C₂₋₁₀alkynyl, or —C₆₋₁₀ aryl; wherein each R^(1A),R^(1B), R^(2A), and R^(2B) is independently selected from —H,—C₁₋₁₀alkyl, —C₂₋₁₀alkenyl, —C₂₋₁₀alkynyl, or —C₆₋₁₀aryl; optionally,wherein R^(1A) and R^(2A) together form an alkylene or heteroalkylenechain of 2-4 atoms in length; optionally, wherein R^(1A) and R^(1B),together with the atom to which they are bound, form a 5-8 memberedheterocyclic substituent moiety, selected from the group consisting ofN-pyrrolidinyl, N-piperidinyl, N-azepanyl, or N-azocanyl; optionally,wherein R^(2A) and R^(2B), together with the atom to which they arebound, form a 5-8 membered heterocyclic substituent moiety, selectedfrom the group consisting of N-pyrrolidinyl, N-piperidinyl, N-azepanyl,or N-azocanyl; and R³ is selected from —C₁₋₁₀alkyl, —C₂₋₁₀alkenyl,—C₂₋₁₀alkynyl, or —C₆₋₁₀aryl, wherein, R^(Si) ₃ is an alkyl or arylgroup, wherein the linker is selected from the group consisting ofsuccinyl, diglycolyl, oxalyl, hydroquinone-O,O′-diacetyl (Q-linker),phthaloyl, 4,5-dichlorophthaloyl, malonyl, glutaryl, diisopropylsilyland 1,1,3,3-tetraisopropyldisiloxane-1,3-diyl, ordodecane-12-oxy-1-phosphatidyl, the label is fluorescein-derivedsubstituent, the quencher is2-((2-hydroxy-ethyl)-{4-[2-methoxy-5-methyl-4-(4-methyl-2-nitro-phenylazo)-phenylazo]-phenyl}-amino)-ethanol-derivedsubstituent; and wherein R^(N) is —H, —C₁₋₄alkyl.
 14. The method ofclaim 13, wherein the phosphorous acid derivative is a phosphitetriester or H-phosphonate diester.
 15. The method of claim 13, whereinthe oxidant is iodine (I₂), bromine (Br₂), chlorine (Cl₂), iodinechloride (ICl), N-bromosuccinimide, N-chlorosuccinimide,N-iodosuccinimide, carbon tetrachloride (CCl₄), bromotrichloromethane(CCl₃Br), tetrabromomethane (CBr₄), tetraiodomethane (CI₄), or iodoform(CHI₃).
 16. The method of claim 13, wherein the oxidant is iodine (I₂).17. The method of claim 13, further comprising a silylating agent and/ora base.
 18. The method of claim 17, wherein the silylating agent isN,O-bis(trimethylsilyl)acetamide (BSA),N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA),chlorotrimethylsilane, bromotrimethylsilane, iodotrimethylsilane,triethylsilyl chloride, triphenylsilyl chloride, hexamethyldisilazane,trimethylsilyl trifluoromethanesulfonate (TMSOTf),dimethylisopropylsilyl chloride, diethylisopropylsilyl chloride,TERT-butyldimethylsilyl chloride, TERT-butyldiphenylsilyl chloride,triisopropylsilyl chloride, dimethyldichlorosilane ordiphenyldichlorosilane.
 19. The method of claim 17, wherein the base istriethylamine, N,N-diisopropylethylamine (DIEA), N-methylmorpholine,N-ethylmorpholine, tributylamine, 1,4-diazabicyclo[2.2.2]octane (DABCO),N-methylimidazole (NMI), pyridine, 2,6-lutidine, 2,4,6-collidine,4-dimethylaminopyridine (DMAP), 1,8-bis(dimethylamino)naphthalene(“proton sponge”), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU),1,5-diazabicyclo[4.3.0]non-5-ene (DBN), 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD), 7-methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene (MTBD),1,1,3,3-tetramethylguanidine (TMG),2-tert-butyl-1,1,3,3-tetramethylguanidine,2,8,9-trimethyl-2,5,8,9-tetraaza-1-phosphabicyclo[3.3.3]undecane or aphosphazene base.
 20. A method of synthesising a compound of Formula(I), the method comprising reaction of a phosphorous acid derivative of

wherein Z is a protecting group (PG); X is selected from the 5′-O— of anucleoside oligonucleotide and Y is selected from the 3′-O— of anucleoside, oligonucleotide; —H, —OH, —SH, —NHR^(N), —O-PG, or —S-PG,wherein PG is a protecting group; a linker, a monophosphate ordiphosphate, or a label or quencher; or Y is selected from the 3′-O— ofa nucleoside, oligonucleotide and X is selected from the 5′-O— of anucleoside, oligonucleotide, —H, —OH, —SH, —NHR^(N), —O-PG, or —S-PG,wherein PG is a protecting group; a linker, a monophosphate ordiphosphate, or a label or quencher, with an organic azide, wherein thelinker is selected from the group consisting of succinyl, diglycolyl,oxalyl, hydroquinone-O,O′-diacetyl (Q-linker), phthaloyl,4,5-dichlorophthaloyl, malonyl, glutaryl, diisopropylsilyl and1,1,3,3-tetraisopropyldisiloxane-1,3-diyl, ordodecane-12-oxy-1-phosphatidyl, the label is fluorescein-derivedsubstituent, the quencher is2-((2-Hydroxy-ethyl)-{4-[2-methoxy-5-methyl-4-(4-methyl-2-nitro-phenylazo)-phenylazo]-phenyl}-amino)-ethanol-derivedsubstituent; and wherein R^(N) is —H, —C₁₋₄alkyl.
 21. The method ofclaim 20, wherein the organic azide comprises of formula:

wherein each R^(1A), R^(1B), R^(2A), and R^(2B) is independently —H,—C₁₋₁₀alkyl, —C₂₋₁₀alkenyl, —C₂₋₁₀alkynyl, or —C₆₋₁₀aryl; and/oroptionally, wherein R^(1A) and R^(2B) together with the atom to whichthey are bound, form a 5-8 membered heterocyclic substituent moiety,selected from the group consisting of N-pyrrolidinyl, N-piperidinyl,N-azepanyl, or N-azocanyl; or optionally, wherein R^(2A) and R^(2B),together with the atom to which they are bound, form a 5-8 memberedheterocyclic substituent moiety, selected from the group consisting ofN-pyrrolidinyl, N-piperidinyl, N-azepanyl, or N-azocanyl; or

wherein each R^(1A) and R^(1B) is independently —H, —C₁₋₁₀alkyl,—C₂₋₁₀alkenyl, —C₂₋₁₀alkynyl, or —C₆₋₁₀aryl; or R^(1A) and R^(1B),together with the atom to which they are bound, form a 5-8 memberedheterocyclic substituent moiety, selected from the group consisting ofN-pyrrolidinyl, N-piperidinyl, N-azepanyl, or N-azocanyl, and R² isselected from —H, —F, —OPG, —Br, —I, —CN, —N₃, —O—C₁₋₁₀alkyl, —SPG, and—S—C₁₋₁₀alkyl, wherein PG is a protecting group, wherein the linker isselected from the group consisting of succinyl, diglycolyl, oxalyl,hydroquinone-O,O′-diacetyl (Q-linker), phthaloyl, 4,5-dichlorophthaloyl,malonyl, glutaryl, diisopropylsilyl and1,1,3,3-tetraisopropyldisiloxane-1,3-diyl, ordodecane-12-oxy-1-phosphatidyl, the label is fluorescein-derivedsubstituent, the quencher is2-((2-hydroxy-ethyl)-{4-[2-methoxy-5-methyl-4-(4-methyl-2-nitro-phenylazo)-phenylazo]-phenyl}-amino)-ethanol-derivedsubstituent; and wherein R^(N) is —H, —C₁₋₄alkyl.
 22. The method ofclaim 20, further comprising a silylating agent and/or a base.
 23. Themethod of claim 22, wherein the silylating agent isN,O-bis(trimethylsilyl)acetamide (BSA),N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA),chlorotrimethylsilane, bromotrimethylsilane, iodotrimethylsilane,triethylsilyl chloride, triphenylsilyl chloride, hexamethyldisilazane,trimethylsilyl trifluoromethanesulfonate (TMSOTf),dimethylisopropylsilyl chloride, diethylisopropylsilyl chloride,TERT-butyldimethylsilyl chloride, TERT-butyldiphenylsilyl chloride,triisopropylsilyl chloride, dimethyldichlorosilane ordiphenyldichlorosilane.
 24. The method of claim 22, wherein the base istriethylamine, N,N-diisopropylethylamine (DIEA), N-methylmorpholine,N-ethylmorpholine, tributylamine, 1,4-diazabicyclo[2.2.2]octane (DABCO),N-methylimidazole (NMI), pyridine, 2,6-lutidine, 2,4,6-collidine,4-dimethylaminopyridine (DMAP), 1,8-bis(dimethylamino)naphthalene(“proton sponge”), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU),1,5-diazabicyclo[4.3.0]non-5-ene (DBN),1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD),7-methyl-1,5,7-triazabicyclo[4.4.0] dec-5-ene (MTBD),1,1,3,3-tetramethylguanidine (TMG),2-tert-butyl-1,1,3,3-tetramethylguanidine,2,8,9-trimethyl-2,5,8,9-tetraaza-1-phosphabicyclo[3.3.3]undecane or aphosphazene base.
 25. An oligonucleotide having at least one modifiedphosphate moiety of formula FVII:

wherein ---- indicates a point of attachment and R¹ and R² are asdefined in claim
 1. 26. An oligonucleotide wherein a phosphate linkingadjacent nucleosides comprises a phosphoryl guanidine, phosphorylamidine, phosphoryl isourea, phosphoryl isothiourea, phosphoryl imidate,or phosphoryl imidothioate.